Templated nanoconjugates

ABSTRACT

The present disclosure is directed to compositions comprising templated nanoconjugates and methods of their use.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase of International ApplicationNo. PCT/US2010/055018 filed Nov. 1, 2010, which claims the prioritybenefit under 35 U.S.C. §119(e) of U.S. Provisional Application No.61/256,640, filed Oct. 30, 2009, U.S. Provisional Application No.61/374,550, filed Aug. 17, 2010 and U.S. Provisional Application No.61/386,846, filed Sep. 27, 2010, the disclosures of which areincorporated herein by reference in their entirety.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under Grant NumberW911NF-09-1-0069, awarded by the U.S. Army RDECOM, and Grant Number U54CA119341, awarded by the National Institutes of Health (NCI-CCNE). Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present disclosure is directed to compositions comprising templatednanoconjugates and methods of their use.

BACKGROUND OF THE INVENTION

Nucleic acids that regulate gene expression are widely considered to bepotential therapeutics as well as important tools for gene functionanalysis. The potential of nucleic acid methods lies in their ability toregulate gene pathways by recognizing and binding complementary targetspresent in cells. However, the delivery of nucleic acids into mammaliancells remains a major challenge, as cells are naturally resistant tonucleic acid uptake. Additionally, they have a variety of mechanismsthat degrade and destroy foreign nucleic acids both inside and outsidethe cell. Therefore, the creation of vectors that can non-toxicallypenetrate cellular membranes and deliver programmed nucleic acidswithout the aid of external transfection agents is necessary for theextension of these technologies to therapeutic application.

Polyvalent inorganic nanomaterials are now recognized as potentialtherapeutic agents in vivo, and in some cases are already FDA clearedfor use as diagnostic tools [Rosi et al., Chem. Rev. 105 (4): 1547-1562(2005); Taton et al., Science 289 (5485): 1757-1760 (2000)]. Because thesurface of these particles can be associated with biomolecules through avariety of attachment strategies, they can be engineered through thechoice of their surface ligands, to interact with well-known biologicalsystems and pathways. For example, by modifying gold nanoparticles witha dense shell of duplexed siRNA, it possible to engage RNAi genesilencing in mammalian cells [Giljohann et al., J. Am. Chem. Soc. 131(6): 2072-2073 (2009)]. These particles are particularly effective asRNAi gene regulation agents because they exhibit high cellular uptakewithout transfection agents [Rosi et al., Science 312 (5776): 1027-1030(2006)], lack of acute toxicity, resistance to nuclease degradation[Seferos et al., Nano Lett. 9(1): 308-11 (2009)] and high stability inbiological media. It is important to note that a growing body of worksuggests that the gold nanoparticle-polynucleotide conjugate's abilityto perform these various functions stems solely from the tightly packedarrangement of polynucleotides on the particles' surface [Giljohann etal., Nano Lett. 7 (12): 3818-21 (2007)]. In another example, abiomimetic synthetic high density lipoprotein (HDL) nanoconjugate can beconstructed by modifying gold nanoparticles with a dense shell ofphospholipids and APO1A, which is a biologically relevant protein[Thaxton et al., J. Am. Chem. Soc. 131 (4): 1384-5 (2009)]. HDL is adynamic serum molecule protective against the development ofatherosclerosis and resultant illnesses such as heart disease andstroke. Like biogenic HDL, this synthetic construct is capable ofbinding cholesterol in its hydrophobic phospholipid shell. Importantly,in both of these cases and many others, it is the dense polyvalentarrangement of biological ligands on the surface of inorganicnanoparticles that imparts their unique ability to interact withbiological systems, regardless of their core material.

Although biological-inorganic nanomaterial hybrids possess desirableattributes, such as those used for diagnostics and therapeutics,concerns have arisen over the clearance/persistence and toxicity of thecore material in vivo. Because these concerns are widely recognized aslimitations for the use of nanomaterials in vivo, a universal approachis needed to create soft nanomaterials with tailorable surfacefunctionalities that would maintain the properties of their inorganicnanoparticle bioconjugate counterparts. Attempts have been made toaddress these problems through a number of synthetic strategies, whichincludes micellar structures [Li et al., Nano Lett. 4 (6): 1055-1058(2004); Liu et al., Chem-Eur J 16 (12): 3791-3797 (2010)].

Hollow nanoconjugates have attracted significant interest in recentyears due to their unique chemical, physical, and biological properties,which suggest a wide range of applications in drug/gene delivery [Shu etal., Biomaterials 31: 6039 (2010); Kim et al., Angew. Chem. Int. Ed. 49:4405 (2010); Kasuya et al., In Meth. Enzymol.; Nejat, D., Ed.; AcademicPress: 2009; Vol. Volume 464, p 147], imaging [Sharma et al., ContrastMedia Mol. Imaging 5: 59 (2010); Tan et al., J. Chem. Commun. 6240(2009)], and catalysis [Choi et al., Chem. Phys. 120: 18 (2010)].Accordingly, a variety of methods have been developed to synthesizethese structures based upon emulsion polymerizations [Anton et al., J.Controlled Release 128: 185 (2008); Landfester et al., J. Polym. Sci.Part A: Polym. Chem. 48: 493 (2010); Li et al., J. Am. Chem. Soc. 132:7823 (2010)], layer-by-layer processes [Kondo et al., J. Am. Chem. Soc.132: 8236 (2010)], crosslinking of micelles [Turner et al., Nano Lett.4: 683 (2004); Sugihara et al., Angew. Chem. Int. Ed. 49: 3500 (2010);Moughton et al., Soft Matter 5: 2361 (2009)], molecular or nanoparticleself-assembly [Kim et al., Angew. Chem. Int. Ed. 46: 3471 (2007); Kim etal., J. Am. Chem. Soc. 132(28): 9908-19 (2010)], and sacrificialtemplate techniques [Réthoré et al., Small 6: 488 (2010)]. Among them,the templating method is particularly powerful in that it transfers theability to control the size and shape of the template to the product,for which desired homogeneity and morphology can be otherwise difficultto achieve. In a typical templated synthesis, a sacrificial core ischosen, upon which preferred materials containing latent crosslinkingmoieties are coated. Following the stabilization of the coating throughchemical crosslinking, the template is removed, leaving the desiredhollow nanoparticle. This additional crosslinking step can be easilyachieved for compositionally simple molecules, such as poly(acrylicacid) or chitosan [Cheng et al., J. Am. Chem. Soc. 128: 6808 (2006); Huet al., Biomacromolecules 8: 1069 (2007)]. However, for systemscontaining sensitive and/or biologically functional structures,conventional crosslinking chemistries may not be sufficiently orthogonalto prevent the loss of their activity.

SUMMARY OF THE INVENTION

The present disclosure provides compositions comprising templatednanoconjugates and methods of their use. Disclosed herein are methodsfor creating nanoconjugates, with or without a surface. In one aspect,biomolecules bearing one or more moieties that can be crosslinked areactivated, which initiates crosslinking reactions between biomolecules.In some aspects, the activation is spontaneous. After the reaction iscomplete, the surface is optionally be dissolved and the resultinghollow structures retain the shape of the surface. The hollow structureswithout the surface exhibit properties of the nanoconjugate in which thesurface remains intact.

Thus, the disclosure provides a plurality of nanoconjugates, eachnanoconjugate having a defined structure and comprising a plurality ofcrosslinked biomolecules in a monolayer, a surface providing a templateupon which the structure is assembled, wherein the surface is optionallyat least partially removed after the structure has been defined.

In various aspects, the nanoparticle is selected from the groupconsisting of a gold nanoparticle, a silver nanoparticle, a platinumnanoparticle, an aluminum nanoparticle, a palladium nanoparticle, acopper nanoparticle, a cobalt nanoparticle, an indium nanoparticle, anda nickel nanoparticle.

In an embodiment, the disclosure provides a nanoconjugate wherein eachbiomolecule in the plurality of biomolecules is the same. In anotherembodiment, the disclosure provides a nanoconjugate wherein at least twoof the biomolecules in the plurality of biomolecules are different.

In various embodiments, the biomolecule is selected from the groupconsisting of a polynucleotide, peptide, polypeptide, phospholipid,oligosaccharide, small molecule, therapeutic agent, contrast agent andcombinations thereof.

In further embodiments, the plurality of nanoconjugates is monodisperse.In various aspects, the monodispersity is such that there is about 25%variation in the diameter of the plurality of nanoconjugates, or whereinthe monodispersity is such that there is about 10% variation in thediameter of the plurality of nanoconjugates, or wherein themonodispersity is such that there is about 1% variation in the diameterof the plurality of nanoconjugates.

In some embodiments, the density of crosslinked biomolecules on thesurface is sufficient for cooperative behavior between the biomolecules.In various aspects, a plurality of nanoconjugates are provided whereindensity of crosslinked biomolecules on the surface is about 2 pmol/cm²,or wherein density of crosslinked biomolecules on the surface is about100 pmol/cm².

In a further aspect of the disclosure, a plurality of nanoconjugates isprovided wherein the plurality of nanoconjugates further comprises anadditional agent. In various aspects, the additional agent is selectedfrom the group consisting of a polynucleotide, peptide, polypeptide,phospholipid, oligosaccharide, metal complex, small molecule,therapeutic agent, contrast agent and combinations thereof.

In one embodiment, the additional agent is associated with at least onebiomolecule of the plurality of biomolecules. In various aspects, theadditional agent is associated with at least one biomolecule of theplurality of biomolecules through hybridization, while in additionalaspects the additional agents is covalently or noncovalently associatedwith at least one biomolecule of the plurality of biomolecules. In afurther aspect, the additional molecule is entrapped in the crosslinkedbiomolecules of at least one of the plurality of nanoconjugates.

The disclosure also provides, in various embodiments, a plurality ofnanoconjugates wherein at least one nanoconjugate in the plurality ofnanoconjugates is hollow in the absence of the surface, or wherein amajority of the nanoconjugates in the plurality of nanoconjugates ishollow in the absence of the surface, or wherein substantially all ofthe nanoconjugates in the plurality of nanoconjugates are hollow in theabsence of the surface.

In one embodiment, a plurality of nanoconjugates is provided wherein inat least one of the plurality of nanoconjugates, an additional agent isencapsulated in the nanoconjugate which is otherwise hollow.

Also provided by the disclosure, in one embodiment, is a method ofcrosslinking a structured nanoconjugate, the method comprising the stepof activating a first biomolecule by contacting the first biomoleculewith a surface, the activation allowing the first biomolecule tocrosslink to a second biomolecule. In one aspect, the surface provides atemplate upon which the structure is assembled, while in another aspectthe surface is a nanoparticle. In various aspects, the nanoparticle isselected from the group consisting of a sphere, a rod and a prism.

The methods provided by the disclosure also provide, in various aspects,that the nanoparticle is metallic, and in further aspects that thenanoparticle is a colloidal metal. Thus, in still further aspects, thenanoparticle is selected from the group consisting of a goldnanoparticle, a silver nanoparticle, a platinum nanoparticle, analuminum nanoparticle, a palladium nanoparticle, a copper nanoparticle,a cobalt nanoparticle, an indium nanoparticle, and a nickelnanoparticle.

The disclosure also provides that in various embodiments of any of themethods described herein, the surface is at least partially removedafter the crosslinking.

Further provided by the disclosure, and in some aspects, are methodswherein the first biomolecule and the second biomolecule are selectedfrom the group consisting of a polynucleotide, peptide, polypeptide,phospholipid, oligosaccharide, small molecule, therapeutic agent,contrast agent and combinations thereof. In further aspects, the firstbiomolecule and the second biomolecule comprise at least one alkynemoiety, or the first biomolecule and the second biomolecule eachcomprise about 10 alkyne moieties.

Regarding the alkyne moiety, the disclosure provides in various aspectsthat the alkyne moiety is activated upon contact with the surface. Inone aspect, the activation renders the alkyne susceptible to anucleophile, and in various embodiments the nucleophile is selected fromthe group consisting of water, an alcohol, an amine, a thiol, an ester,a thioester, urea, an amide, an aldehyde, a carbonate, a carbamate, anintramolecular hydroxyl group, a methyl ether group, a benzylic ethergroup, a carboxylic acid, a ketone, an imine, phenol, 2-pyrrolidone, anindole, acetic acid, a β-ketoester and combinations thereof.

In various embodiments of the disclosure, activation causes crosslinkingof the first biomolecule to the second biomolecule.

In some aspects, the first biomolecule is a polynucleotide, and infurther aspects the second biomolecule is a polynucleotide. In stillfurther aspects, the polynucleotide is a DNA polynucleotide or a RNApolynucleotide.

The disclosure also provides, in various embodiments, that thepolynucleotide is about 5 to about 100 nucleotides in length, about 5 toabout 90 nucleotides in length. about 5 to about 80 nucleotides inlength, about 5 to about 70 nucleotides in length, about 5 to about 60nucleotides in length, about 5 to about 50 nucleotides in length, about5 to about 45 nucleotides in length, about 5 to about 40 nucleotides inlength, about 5 to about 35 nucleotides in length, about 5 to about 30nucleotides in length, about 5 to about 25 nucleotides in length, about5 to about 20 nucleotides in length, about 5 to about 15 nucleotides inlength, or about 5 to about 10 nucleotides in length.

In another embodiment, the additional agent is added to the firstbiomolecule and the second biomolecule during the crosslinking step,while in another embodiment the additional agent is added to thenanoconjugate after formation of the nanoconjugate but before removal ofthe surface. In a further embodiment, the additional agent is added tothe nanoconjugate after formation of the nanoconjugate and after removalof the surface.

In another aspect, a composition is provided comprising a polyvalentnanoconjugate comprising a surface, the nanoconjugate further comprisinga plurality of polynucleotides wherein a spacer end of each of thepolynucleotides in the plurality is modified such that contacting theplurality of polynucleotides with a chemical crosslinks the plurality ofpolynucleotides, wherein the surface is optionally at least partiallyremoved after the crosslinking. In various aspects of the composition,the nanoconjugate comprises a nanoparticle, and in further aspects thenanoparticle is selected from the group consisting of a sphere, a rodand a prism.

Accordingly, in still further aspects of the composition, thenanoparticle is metallic. In one aspect, the nanoparticle is a colloidalmetal. In various embodiments of the composition, the nanoparticle isselected from the group consisting of a gold nanoparticle, a silvernanoparticle, a platinum nanoparticle, an aluminum nanoparticle, apalladium nanoparticle, a copper nanoparticle, a cobalt nanoparticle, anindium nanoparticle, and a nickel nanoparticle.

In various embodiments, the modification is selected from the groupconsisting of an amine, amide, alcohol, ester, aldehyde, ketone, thiol,disulfide, carboxylic acid, phenol, imidazole, hydrazine, hydrazone,azide and an alkyne. In one aspect, the modification is anamine-modified nucleotide, and in a further aspect the amine-modifiedphosphoramidite nucleotide is an amine-modified thymidinephosphoramidite (TN).

In further embodiments, the chemical is selected from the groupconsisting of Disuccinimidyl glutarate, Disuccinimidyl suberate,Bis[sulfosuccinimidyl] suberate, Tris-succinimidyl aminotriacetate,succinimidyl 4-hydrazinonicotinate acetone hydrazone, succinimidyl4-hydrazidoterephthalate hydrochloride, succinimidyl 4-formylbenzoate,Dithiobis[succinimidyl propionate],3,3′-Dithiobis[sulfosuccinimidylpropionate], Disuccinimidyl tartarate,Bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone, Ethylene glycolbis[succinimidylsuccinate], Ethylene glycolbis[sulfosuccinimidylsuccinate], Dimethyl adipimidate.2 HCl, Dimethylpimelimidate.2 HCl, Dimethyl Suberimidate.2 HCl,1,5-Difluoro-2,4-dinitrobenzene,β-[Tris(hydroxymethyl)phosphino]propionic acid, Bis-Maleimidoethane,1,4-bismaleimidobutane, Bismaleimidohexane, Tris[2-maleimidoethyl]amine,1,8-Bis-maleimido-diethyleneglycol,1,11-Bis-maleimido-triethyleneglycol, 1,4bismaleimidyl-2,3-dihydroxybutane, Dithio-bismaleimidoethane,1,4-Di-[3′-(2′-pyridyldithio)-propionamido]butane,1,6-Hexane-bis-vinylsulfone,Bis-[b-(4-Azidosalicylamido)ethyl]disulfide, N-(a-Maleimidoacetoxy)succinimide ester, N-[β-Maleimidopropyloxy]succinimide ester,N[g-Maleimidobutyryloxy]succinimide ester,N-[g-Maleimidobutyryloxy]sulfosuccinimide ester,m-Maleimidobenzoyl-N-hydroxysuccinimide ester,m-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester, Succinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate, Sulfosuccinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate,N-e-Maleimidocaproyloxy]succinimide ester,N-e-Maleimidocaproyloxy]sulfosuccinimide ester, Succinimidyl4-[p-maleimidophenyl]butyrate, Sulfosuccinimidyl4-[p-maleimidophenyl]butyrate,Succinimidyl-6-[β-maleimidopropionamido]hexanoate,Succinimidyl-4-[N-Maleimidomethyl]cyclohexane-1-carboxy-[6-amidocaproate],N-[k-Maleimidoundecanoyloxy]sulfosuccinimide ester, N-Succinimidyl3-(2-pyridyldithio)-propionate, Succinimidyl6-(3-[2-pyridyldithio]-propionamido)hexanoate,4-Succinimidyloxycarbonyl-methyl-a-[2-pyridyldithio]toluene,4-Sulfosuccinimidyl-6-methyl-a-(2-pyridyldithio)toluamido]hexanoate),N-Succinimidyl iodoacetate, Succinimidyl 3-[bromoacetamido]propionate,N-Succinimidyl[4-iodoacetyl]aminobenzoate,N-Sulfosuccinimidyl[4-iodoacetyl]aminobenzoate,N-Hydroxysuccinimidyl-4-azidosalicylic acid,N-5-Azido-2-nitrobenzoyloxysuccinimide,N-Hydroxysulfosuccinimidyl-4-azidobenzoate,Sulfosuccinimidyl[4-azidosalicylamido]-hexanoate,N-Succinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate,N-Sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate,Sulfosuccinimidyl-(perfluoroazidobenzamido)-ethyl-1,3′-dithioproprionate,Sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3′-proprionate,Sulfosuccinimidyl2-[7-amino-4-methylcoumarin-3-acetamido]ethyl-1,3′dithiopropionate,Succinimidyl 4,4′-azipentanoate, Succinimidyl6-(4,4′-azipentanamido)hexanoate, Succinimidyl2-([4,4′-azipentanamido]ethyl)-1,3′-dithioproprionate, Sulfosuccinimidyl4,4′-azipentanoate, Sulfosuccinimidyl 6-(4,4′-azipentanamido)hexanoate,Sulfosuccinimidyl 2-([4,4′-azipentanamido]ethyl)-1,3′-dithioproprionate,Dicyclohexylcarbodiimide, 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimideHydrochloride, N-[4-(p-Azidosalicylamido)butyl]-3′-(2′-pyridyldithio)propionamide, N-[β-Maleimidopropionicacid]hydrazide, trifluoroacetic acid salt, [N-e-Maleimidocaproicacid]hydrazide, trifluoroacetic acid salt,4-(4-N-Maleimidophenyl)butyric acid hydrazide hydrochloride,N-[k-Maleimidoundecanoic acid]hydrazide, 3-(2-Pyridyldithio)propionylhydrazide, p-Azidobenzoyl hydrazide, N-[p-Maleimidophenyl]isocyanate andSuccinimidyl-[4-(psoralen-8-yloxy)]-butyrate.

In another aspect, the plurality of polynucleotides comprise DNApolynucleotides, RNA polynucleotides or a combination thereof, and invarious embodiments the polynucleotide is about 5 to about 100nucleotides in length, about 5 to about 90 nucleotides in length. about5 to about 80 nucleotides in length, about 5 to about 70 nucleotides inlength, about 5 to about 60 nucleotides in length, about 5 to about 50nucleotides in length, about 5 to about 45 nucleotides in length, about5 to about 40 nucleotides in length, about 5 to about 35 nucleotides inlength, about 5 to about 30 nucleotides in length, about 5 to about 25nucleotides in length, about 5 to about 20 nucleotides in length, about5 to about 15 nucleotides in length, or about 5 to about 10 nucleotidesin length.

In further embodiments, the disclosure also provides methods of makingany of the nanoconjugates of the disclosure using a method describedherein.

In another aspect of the disclosure a method of detecting a targetmolecule is provided comprising contacting the target molecule with anyof the nanoconjugate compositions described herein, wherein contactbetween the target molecule and the composition results in a detectablechange. In various aspects, the detecting is in vitro or the detectingis in vivo.

The disclosure also provides, in various aspects, a method of inhibitingexpression of a gene product encoded by a target polynucleotidecomprising contacting the target polynucleotide with any of thenanoconjugate compositions described herein under conditions sufficientto inhibit expression of the gene product. In various aspects,expression of the gene product is inhibited in vivo or expression of thegene product is inhibited in vitro. In a further aspect, expression ofthe gene product is inhibited by at least about 5%.

The disclosure additionally provides, in various embodiments,compositions of the disclosure made by any of the methods describedherein.

DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts modification of amine-modified DNA with an alkyne-NHSester.

FIG. 2 shows A) Dissolution of multiple citrate stabilized, DNAfunctionalized, and alkyne-DNA functionalized AuNPs. B) Alkyne-DNAfunctionalized AuNPs at various stages in the dissolution process. C)UV-Vis spectra of the corresponding time points in B.

FIG. 3 shows TEM images of the indicated solutions of gold nanoparticlesduring the dissolution process.

FIG. 4 shows A) Gel electrophoresis comparison of free strand DNA tocrosslinked structures and DNA functionalized AuNPs. B) Gelelectrophoresis analysis of crosslinked structures obtained fromalkyne-DNA-AuNPs with a range of densities.

FIG. 5 depicts A) Gel electrophoresis of hollow DNA nanoconjugatesformed from a range of different sized templates. B) Gel electrophoresisof crosslinked structures obtained from AuNPs functionalized withalkyne-DNA that were modified with varying numbers of alkyne units.

FIG. 6 shows the two strand system of Fl and Cy3 modified hollow DNAnanoconjugates. At 60° C., these particles were dehybridized and thegreen fluorescence of fluorescein was observed. At room temperature, theparticles hybridized and FRET from fluorescein to Cy3 produced theorange fluorescence of Cy3. After time, these particles form macroscopicaggregates that settle out of solution.

FIG. 7 depicts extinction as a function of temperature for free strandDNA at 260 nm. DNA-AuNP aggregates formed from these strands at 520 nmand hollow DNA nanoconjugate aggregates at 260 nm formed from the samestrands.

FIG. 8 depicts A) RT-qPCR quantification of EGFR mRNA for cells treatedwith dharmafect (free), siRNA-AuNPs and siRNA-hollow nanoparticlesnormalized to GAPDH. B) Western blot for EGFR and GAPDH in cells treatedwith siRNA-hollow nanoparticles.

FIG. 9 shows A) Dissolution process of polymer-coated 13 nm AuNP. B)Normalized UV-Vis spectra of AuNP at various time points during thedissolution. C) TEM images of a) polymer-coated AuNP, b-c) partiallyformed nanoconjugates and d) fully formed nanoconjugates (negativelystained with 0.5% uranyl acetate).

FIG. 10 shows A.) Number-average hydrodynamic diameters of AuNPs (13,20, 30 and 40 nm), polymer-coated AuNPs and polymer nanoconjugates(PNSs) measured by dynamic light scattering. B.) TEM images of a) 20 nmPNSs and b) 40 nm PNSs (negatively stained with 0.5% uranyl acetate). Inc) it is illustrated why nanocapsules appear as donut-shaped in TEM.Black: uranyl acetate stain. Blue: a collapsed PNS on a surface.

FIG. 11 shows A.) 1H-13C NMR spectrum of 1. B.) 1H-13C NMR spectrum of4. C.) 1H-1H COSY of 4. D.) IR spectrum of 1 (dashed line) and 4 (solidline).

FIG. 12 shows A) HSQC for 5. B) HSQC for 5 after reaction. C) MALDI-TOFMS for 5 before and after reaction.

FIG. 13 shows A) MALDI-TOF of DNA-propargyl ether conjugate 6 afterincubation with AuNP. B) Agarose gel (3%) electrophoresis of 6 afterincubation with AuNP of 6, confirming the prediction of the proposedcrosslinking mechanism that a 3-arm crosslink is possible with a singleacetylene group (FIG. 12).

FIG. 14A depicts the construction of proteonanoconjugates containingstreptavidin and HRP. FIG. 14B depicts a surface-based chromogenicanalysis of the HRP activity of the proteonanoconjugate.

FIG. 15 shows HRP-catalyzed chromogenic analysis ofproteonanoconjugates, showing successful incorporation of streptavidinand HRP into the nanoconjugate shell and retention of the proteinfunction.

FIG. 16 depicts a potential pathway for formation of hollow HDLnanoparticles by using an alkyne moiety and a phospholipid-bearingpolymer and APO1A proteins.

FIG. 17 depicts a potential pathway for poly alkyne crosslinking.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are compositions comprising a plurality ofnanoconjugates, each nanoconjugate having a defined structure andcomprising a plurality of crosslinked biomolecules in a monolayer, thestructure defined by a surface and the plurality of nanoconjugates beingmonodisperse, wherein the presence of the surface is optional after thestructure has been defined.

The nanoconjugates disclosed are effective alternatives tofunctionalized nanoparticles as described in, for example, Rosi et al.,Chem. Rev. 105(4): 1547-1562 (2005), Taton et al., Science 289(5485):1757-1760 (2000), PCT/US2006/022325 and U.S. Pat. No. 6,361,944 becausethey exhibit high cellular uptake without transfection agents, lackacute toxicity, exhibit resistance to nuclease degradation and have highstability in biological media. The combination of these properties issignificant since, despite the tremendously high uptake ofbiomolecule-functionalized nanoparticles, they exhibit no toxicity inthe cell types tested thus far (see Table 1, below), and this propertyis critical for therapeutic agent delivery applications for reducingoff-target effects.

TABLE 1 Cell Type Designation or Source Breast SKBR3, MDA-MB-321, AU-565Brain U87, LN229 Bladder HT-1376, 5637, T24 Colon LS513 Cervix HeLa,SiHa Skin C166, KB, MCF, 10A Kidney MDCK Blood Sup T1, Jurkat LeukemiaK562 Liver HepG2 Kidney 293T Ovary CHO Macrophage RAW 264.7 HippocampusNeurons primary, rat Astrocytes primary, rat Glial Cells primary, ratBladder primary, human Erythrocytes primary, mouse Peripheral Bloodprimary, mouse Mononuclear Cell T-Cells primary, human Beta Isletsprimary, mouse Skin primary, mouse

The disclosure thus provides compositions and methods relating to thegeneration of nanoconjugates. In one aspect, the nanoconjugate comprisesbiomolecules that are crosslinked to each other and attached to asurface. In some aspects, the surface is dissolved leaving a hollownanoconjugate. Thus, a nanoconjugate comprising a biomolecule and/ornon-biomolecule as used herein can will be understood to mean either ananoconjugate in which the surface is retained, or a nanoconjugate inwhich the surface has been dissolved as described herein. Ananoconjugate in which the surface has been dissolved is referred toherein as a hollow nanoconjugate or hollow particle.

Accordingly, a plurality of nanoconjugates is provided wherein eachnanoconjugate has a defined structure and comprises a plurality ofcrosslinked biomolecules in a monolayer. The structure of eachnanoconjugate is defined by (i) the surface that was used in themanufacture of the nanoconjugates (ii) the type of biomolecules formingthe nanoconjugate, and (iii) the degree and type of crosslinking betweenindividual biomolecules on and/or around the surface. While the surfaceis integral for producing the nanoconjugates, the surface is notintegral to maintaining the structure of the nanoconjugates. Thus inalternative embodiments, the plurality of nanoconjugates either includesthe surface used in their production or the plurality includes a partialsurface, or the plurality does not include the surface.

The term “surface” means the structure on or around which thenanoconjugate forms.

As used herein, a “biomolecule” is understood to include apolynucleotide, peptide, polypeptide, phospholipid, oligosaccharide,small molecule, therapeutic agent, contrast agent and combinationsthereof.

It is noted here that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referenceunless the context clearly dictates otherwise.

It is also noted that the term “about” as used herein is understood tomean approximately.

It is further noted that the terms “attached,” “conjugated” and“functionalized” are used interchangeably herein and refer to theassociation of a polynucleotide, peptide, polypeptide, phospholipid,oligosaccharide, metal complex, small molecule, therapeutic agent,contrast agent and combinations thereof with a surface.

As used herein, a “majority” means greater than 50% of a population (forexample and without limitation, a population of biomolecules or apopulation of nanoconjugates). Also as used herein, “substantially all”means 90% or greater of a population.

Nanoconjugates Biomolecules/Non-Biomolecules

The basic component of nanoconjugates provided is a plurality ofbiomolecules. In alternative embodiments, however, nanoconjugates areprovided wherein one or more non-biomolecules are included in theplurality of biomolecules. Because of the methods of production, theresulting nanoconjugates are at most a monolayer of biomolecules ormixture of biomolecules and non-biomolecules. As used herein, a“monolayer” means that only a single stratum of biomolecules and/ornon-biomolecules is crosslinked at the surface of a nanoconjugate.

A biomolecule as used herein includes without limitation apolynucleotide, peptide, polypeptide, phospholipid, oligosaccharide,small molecule, therapeutic agent, contrast agent and combinationsthereof.

A non-biomolecule as used herein includes without limitation a diluentmolecule, a metal complex and any non-carbon containing molecule knownin the art.

In various aspects of the nanoconjugate, all of the biomolecules areidentical, or in the alternative, at least two biomolecules aredifferent. Likewise, when a non-biomolecule is included, in one aspectall of the non-biomolecules are identical, and in another aspect atleast two of the non-biomolecules are different. Combinations, whereinall biomolecules are identical and all non-biomolecules are identicalare contemplated, along with mixtures wherein at least two biomoleculesare combined with non-biomolecules that are all identical, allbiomolecules are identical and at least two non-biomolecules aredifferent, and at least two different biomolecules are combined with atleast two different non-biomolecules.

A biomolecule and/or non-biomolecule as used herein will be understoodto either be a structural biomolecule and/or structural non-biomoleculethat are integral to the nanoconjugate structure, or a non-structuralbiomolecule and/or non-structural non-biomolecule that are not integralto the nanoconjugate structure. In some aspects wherein the biomoleculeand/or non-biomolecule is non-structural, the biomolecule and/ornon-biomolecule do not contain a crosslinking moiety. In thisdisclosure, non-structural biomolecules and non-structuralnon-biomolecules are referred to as additional agents.

Structure

The “structure” of a nanoconjugate is understood to be defined, invarious aspects, by (i) the surface that was used in the manufacture ofthe nanoconjugates (ii) the type of biomolecules forming thenanoconjugate, and/or (iii) the degree and type of crosslinking betweenindividual biomolecules on and/or around the surface.

In every aspect of the nanoconjugate provided, the biomolecules, with orwithout a non-biomolecule, are crosslinked. Crosslinking betweenbiomolecules, with or without a non-biomolecule, is effected at a moietyon each biomolecule that can crosslink. If the nanoconjugate includesboth biomolecules and non-biomolecules as structural components, thebiomolecules and non-biomolecules are in some aspects conjugatedtogether. It will be appreciated that some degree of intramolecularcrosslinking may arise in formation of a nanoconjugate in instanceswherein a biomolecule and/or non-biomolecule includes multiplecrosslinking moieties.

In some aspects, a crosslinking moiety is a moiety that can becomeactivated to crosslink. An activated moiety means that the crosslinkingmoiety present on a biomolecule, and/or non-biomolecule when present, isin a state that makes the moiety able to crosslink to anotherbiomolecule, and/or non-biomolecule when present, that also contains acrosslinking moiety. The crosslinking moieties on a plurality ofbiomolecules and/or non-biomolecules can be the same for everybiomolecule and/or non-biomolecules in the plurality, or at least twobiomolecules and/or non-biomolecules in the plurality can containdifferent crosslinking moieties. A single biomolecule and/ornon-biomolecule is also contemplated to comprise more than onecrosslinking moiety, and those moieties can be the same or different.

In one aspect, the crosslinking moiety is located in the same positionin each biomolecule, and/or non-biomolecule when present, which undercertain conditions orients all of the biomolecules, andnon-biomolecules, in the same direction.

In another aspect, the crosslinking moiety is located in differentpositions in the biomolecules, and/or non-biomolecules, which undercertain conditions can provide mixed orientation of the biomoleculesafter crosslinking.

Shape

The shape of each nanoconjugate in the plurality is determined by thesurface used in its production, and optionally by the biomoleculesand/or non-biomolecules used in its production as well as well thedegree and type of crosslinking between and among the biomoleculesand/or non-biomolecules. The surface is in various aspects planar orthree dimensional. Necessarily a planar surface will give rise to aplanar nanoconjugate and a three dimensional surface will give rise to athree dimensional shape that mimics the three dimensional surface. Whenthe surface is removed, a nanoconjugate formed with a planar surfacewill still be planar, and a nanoconjugate formed with a threedimensional surface will have the shape of the three dimensional surfaceand will be hollow.

Density

Depending on the degree of crosslinking and the amount of startingcomponent, i.e., the biomolecules or mixture of biomolecules andno-biomolecules, in the preparative mixture, the nanoconjugates providedare contemplated to have varying densities. Thus, the surface is, in oneaspect, completely covered with crosslinked biomolecules or crosslinkedmixture of biomolecules and non-biomolecules, or in an alternativeaspects, significantly covered with crosslinked biomolecules orcrosslinked mixture of biomolecules and non-biomolecules, or sparselycovered with the crosslinked biomolecules or crosslinked mixture ofbiomolecules and non-biomolecules. The density of coverage of thesurface is, in one aspect, even over the entire surface, or in thealternative, the density is uneven over the surface.

The density of the crosslinked biomolecules or crosslinked mixture ofbiomolecules and non-biomolecules of the nanoconjugate, and/or theevenness or lack of evenness of the density over the surface willdetermine the porosity of the nanoconjugate. In various aspects, theporosity determines the ability of the nanoconjugate to entrapadditional, non-structural agents, as discussed below, in the interiorof the nanoconjugate after the surface is removed.

Additional Agents

With regard to non-structural components, the nanoconjugates providedoptionally include an additional agent which in one aspect as discussedabove, is entrapped in the interior of a hollow nanoconjugate.Alternatively, the additional agent is embedded, or enmeshed, in thestructural crosslinked biomolecules or mixture of crosslinkedbiomolecules and non-biomolecules or simply associated with one or bothsurfaces of structural crosslinked biomolecules or mixture ofcrosslinked biomolecule and non-biomolecules. It is contemplated thatthis additional agent is in one aspect covalently associated with thenanoconjugate, or in the alternative, non-covalently associated with thenanoconjugate. However, it is understood that the disclosure providesnanoconjugates wherein one or more additional agents are both covalentlyand non-covalently associated with the nanoconjugate. It will also beunderstood that non-covalent associations include hybridization (i.e.,between polynucleotides), protein binding (i.e., between proteins whichcan bind) and/or hydrophobic interactions (i.e., between lipids andother agents that include a sufficiently hydrophobic domain). In otheraspects, the additional agent is entrapped within the interior of ahollow nanoconjugate. When the nanoconjugate includes this additionalagent, it is contemplated in one aspect that all of the additionalagents are the same, and in other aspects, at least two of theadditional agents are different.

Additional agents contemplated by the disclosure include withoutlimitation a biomolecule, non-biomolecule, detectable marker, a coating,a polymeric agent, a contrast agent, an embolic agent, a short internalcomplementary polynucleotide (sicPN), a transcriptional regulator, atherapeutic agent, an antibiotic and a targeting moiety. These types ofadditional agents are discussed in detail below.

Biomolecules/Non-Biomolecules

Biomolecules, and non-biomolecules when present, with the ability tocrosslink to other biomolecules and/or non-biomolecules, representstructural components of a nanoconjugate. As noted above, biomolecules,and non-biomolecules are also contemplated as being additional,non-structural agents of a nanoconjugate. As described, biomoleculesinclude a polynucleotide, peptide, polypeptide, phospholipid,oligosaccharide, small molecule, therapeutic agent, contrast agent andcombinations thereof.

Common to all structural biomolecules and/or non-biomolecules of thedisclosure is that they comprise one or more crosslinking moieties.Non-structural biomolecules and/or non-biomolecules are contemplatedthat either do or do not possess crosslinking capability, but thenon-structural biomolecules and/or non-biomolecules that do crosslinkare not integral to maintaining the nanoconjugate structure.

The discussion that follow addresses biomolecules and/ornon-biomolecules which are either structural or non-structural. Asmentioned above, non-structural biomolecules and non-biomolecules asadditional agents are also discussed in detail in a separate sectionbelow.

Polynucleotides

Polynucleotides contemplated by the present disclosure include DNA, RNA,modified forms and combinations thereof as defined herein. Accordingly,in some aspects, the nanoconjugate comprises DNA. In some embodiments,the DNA is double stranded, and in further embodiments the DNA is singlestranded. In further aspects, the nanoconjugate comprises RNA, and instill further aspects the nanoconjugate comprises double stranded RNA,and in a specific embodiment, the double stranded RNA agent is a smallinterfering RNA (siRNA). The term “RNA” includes duplexes of twoseparate strands, as well as single stranded structures. Single strandedRNA also includes RNA with secondary structure. In one aspect, RNAhaving a hairpin loop in contemplated.

When a nanoconjugate comprise a plurality of structural polynucleotidebiomolecules, the polynucleotide is, in some aspects, comprised of asequence that is sufficiently complementary to a target sequence of apolynucleotide such that hybridization of the polynucleotide that ispart of the nanoconjugate and the target polynucleotide takes place. Thepolynucleotide in various aspects is single stranded or double stranded,as long as the double stranded molecule also includes a single strandsequence that hybridizes to a single strand sequence of the targetpolynucleotide. In some aspects, hybridization of the polynucleotidethat is part of the nanoconjugate can form a triplex structure with adouble-stranded target polynucleotide. In another aspect, a triplexstructure can be formed by hybridization of a double-strandedpolynucleotide that is part of a nanoconjugate to a single-strandedtarget polynucleotide. Further description of triplex polynucleotidecomplexes is found in PCT/US2006/40124, which is incorporated herein byreference in its entirety.

In some aspects, polynucleotides contain a spacer as described herein.The spacer, in one aspect, comprises one or more crosslinking moietiesthat facilitate the crosslinking of one polynucleotide to anotherpolynucleotide.

A “polynucleotide” is understood in the art to comprise individuallypolymerized nucleotide subunits. The term “nucleotide” or its plural asused herein is interchangeable with modified forms as discussed hereinand otherwise known in the art. In certain instances, the art uses theterm “nucleobase” which embraces naturally-occurring nucleotide, andnon-naturally-occurring nucleotides which include modified nucleotides.Thus, nucleotide or nucleobase means the naturally occurring nucleobasesadenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U).Non-naturally occurring nucleobases include, for example and withoutlimitations, xanthine, diaminopurine, 8-oxo-N6-methyladenine,7-deazaxanthine, 7-deazaguanine, N4,N4-ethanocytosin,N′,N′-ethano-2,6-diaminopurine, 5-methylcytosine (mC),5-(C3-C6)-alkynyl-cytosine, 5-fluorouracil, 5-bromouracil,pseudoisocytosine, 2-hydroxy-5-methyl-4-tr-iazolopyridin, isocytosine,isoguanine, inosine and the “non-naturally occurring” nucleobasesdescribed in Benner et al., U.S. Pat. No. 5,432,272 and Susan M. Freierand Karl-Heinz Altmann, 1997, Nucleic Acids Research, vol. 25: pp4429-4443. The term “nucleobase” also includes not only the known purineand pyrimidine heterocycles, but also heterocyclic analogues andtautomers thereof. Further naturally and non-naturally occurringnucleobases include those disclosed in U.S. Pat. No. 3,687,808 (Merigan,et al.), in Chapter 15 by Sanghvi, in Antisense Research andApplication, Ed. S. T. Crooke and B. Lebleu, CRC Press, 1993, inEnglisch et al., 1991, Angewandte Chemie, International Edition, 30:613-722 (see especially pages 622 and 623, and in the ConciseEncyclopedia of Polymer Science and Engineering, J. I. Kroschwitz Ed.,John Wiley & Sons, 1990, pages 858-859, Cook, Anti-Cancer Drug Design1991, 6, 585-607, each of which are hereby incorporated by reference intheir entirety). In various aspects, polynucleotides also include one ormore “nucleosidic bases” or “base units” which are a category ofnon-naturally-occurring nucleotides that include compounds such asheterocyclic compounds that can serve like nucleobases, includingcertain “universal bases” that are not nucleosidic bases in the mostclassical sense but serve as nucleosidic bases. Universal bases include3-nitropyrrole, optionally substituted indoles (e.g., 5-nitroindole),and optionally substituted hypoxanthine. Other desirable universal basesinclude, pyrrole, diazole or triazole derivatives, including thoseuniversal bases known in the art.

Modified nucleotides are described in EP 1 072 679 and WO 97/12896, thedisclosures of which are incorporated herein by reference. Modifiednucleotides include without limitation, 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine and other alkynyl derivatives of pyrimidine bases,6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine,8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and3-deazaguanine and 3-deazaadenine. Further modified bases includetricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzox-azin-2(3H)-one),carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindolecytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modifiedbases may also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Additional nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., 1991, Angewandte Chemie, International Edition, 30:613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.,ed., CRC Press, 1993. Certain of these bases are useful for increasingthe binding affinity and include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. and are, in certain aspects combinedwith 2′-O-methoxyethyl sugar modifications. See, U.S. Pat. No.3,687,808, U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273;5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177;5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617;5,645,985; 5,830,653; 5,763,588; 6,005,096; 5,750,692 and 5,681,941, thedisclosures of which are incorporated herein by reference.

Methods of making polynucleotides of a predetermined sequence arewell-known. See, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual (2nd ed. 1989) and F. Eckstein (ed.) Oligonucleotides andAnalogues, 1st Ed. (Oxford University Press, New York, 1991).Solid-phase synthesis methods are preferred for both polyribonucleotidesand polydeoxyribonucleotides (the well-known methods of synthesizing DNAare also useful for synthesizing RNA). Polyribonucleotides can also beprepared enzymatically. Non-naturally occurring nucleobases can beincorporated into the polynucleotide, as well. See, e.g., U.S. Pat. No.7,223,833; Katz, J. Am. Chem. Soc., 74:2238 (1951); Yamane, et al., J.Am. Chem. Soc., 83:2599 (1961); Kosturko, et al., Biochemistry, 13:3949(1974); Thomas, J. Am. Chem. Soc., 76:6032 (1954); Zhang, et al., J. Am.Chem. Soc., 127:74-75 (2005); and Zimmermann, et al., J. Am. Chem. Soc.,124:13684-13685 (2002).

Surfaces provided that are used to template a polynucleotide, or amodified form thereof, generally comprise a polynucleotide from about 5nucleotides to about 100 nucleotides in length. More specifically,nanoconjugates comprise polynucleotides that are about 5 to about 90nucleotides in length, about 5 to about 80 nucleotides in length, about5 to about 70 nucleotides in length, about 5 to about 60 nucleotides inlength, about 5 to about 50 nucleotides in length about 5 to about 45nucleotides in length, about 5 to about 40 nucleotides in length, about5 to about 35 nucleotides in length, about 5 to about 30 nucleotides inlength, about 5 to about 25 nucleotides in length, about 5 to about 20nucleotides in length, about 5 to about 15 nucleotides in length, about5 to about 10 nucleotides in length, and all polynucleotidesintermediate in length of the sizes specifically disclosed to the extentthat the polynucleotide is able to achieve the desired result.Accordingly, polynucleotides of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more nucleotidesin length are contemplated.

Polynucleotides, as defined herein, also includes aptamers. Theproduction and use of aptamers is known to those of ordinary skill inthe art. In general, aptamers are nucleic acid or peptide bindingspecies capable of tightly binding to and discreetly distinguishingtarget ligands [Yan et al., RNA Biol. 6(3) 316-320 (2009), incorporatedby reference herein in its entirety]. Aptamers, in some embodiments, maybe obtained by a technique called the systematic evolution of ligands byexponential enrichment (SELEX) process [Tuerk et al., Science 249:505-10(1990), U.S. Pat. No. 5,270,163, and U.S. Pat. No. 5,637,459, each ofwhich is incorporated herein by reference in their entirety]. Generaldiscussions of nucleic acid aptamers are found in, for example andwithout limitation, Nucleic Acid and Peptide Aptamers: Methods andProtocols (Edited by Mayer, Humana Press, 2009) and Crawford et al.,Briefings in Functional Genomics and Proteomics 2(1): 72-79 (2003).Additional discussion of aptamers, including but not limited toselection of RNA aptamers, selection of DNA aptamers, selection ofaptamers capable of covalently linking to a target protein, use ofmodified aptamer libraries, and the use of aptamers as a diagnosticagent and a therapeutic agent is provided in Kopylov et al., MolecularBiology 34(6): 940-954 (2000) translated from Molekulyarnaya Biologiya,Vol. 34, No. 6, 2000, pp. 1097-1113, which is incorporated herein byreference in its entirety. In various aspects, an aptamer is between10-100 nucleotides in length.

Spacers

In certain aspects, nanoconjugates are contemplated which include thosewherein a nanoconjugate comprises a biomolecule which further comprisesa spacer. The spacer in various aspects comprises one or morecrosslinking moieties as described below.

“Spacer” as used herein means a moiety that serves to contain one ormore crosslinking moieties, or, in some aspects wherein thenanoconjugate comprises a nanoparticle, increase distance between thenanoparticle and the biomolecule, or to increase distance betweenindividual biomolecules when attached to the nanoparticle in multiplecopies. In aspects of the disclosure wherein a nanoconjugate is used fora biological activity, it is contemplated that the spacer does notdirectly participate in the activity of the biomolecule to which it isattached.

Thus, in some aspects, the spacer is contemplated herein to facilitatecrosslinking via one or more crosslinking moieties. Spacers areadditionally contemplated, in various aspects, as being located betweenindividual biomolecules in tandem, whether the biomolecules have thesame sequence or have different sequences. In one aspect, the spacerwhen present is an organic moiety. In another aspect, the spacer is apolymer, including but not limited to a water-soluble polymer, a nucleicacid, a polypeptide, an oligosaccharide, a carbohydrate, a lipid, orcombinations thereof.

In instances wherein the spacer is a polynucleotide, the length of thespacer in various embodiments at least about 5 nucleotides, at leastabout 10 nucleotides, 10-30 nucleotides, or even greater than 30nucleotides. The spacer may have any sequence which does not interferewith the ability of the polynucleotides to become bound to thenanoparticles or to the target polynucleotide. The spacers should nothave sequences complementary to each other or to that of thepolynucleotides, but may be all or in part complementary to the targetpolynucleotide. In certain aspects, the bases of the polynucleotidespacer are all adenines, all thymines, all cytidines, all guanines, alluracils, or all some other modified base.

Modified Polynucleotides

As discussed above, modified polynucleotides are contemplated for use inproducing nanoconjugates, and are template by a surface. In variousaspects, a polynucleotide templated on a surface is completely modifiedor partially modified. Thus, in various aspects, one or more, or all,sugar and/or one or more or all internucleotide linkages of thenucleotide units in the polynucleotide are replaced with “non-naturallyoccurring” groups.

In one aspect, this embodiment contemplates a peptide nucleic acid(PNA). In PNA compounds, the sugar-backbone of a polynucleotide isreplaced with an amide containing backbone. See, for example U.S. Pat.Nos. 5,539,082; 5,714,331; and 5,719,262, and Nielsen et al., Science,1991, 254, 1497-1500, the disclosures of which are herein incorporatedby reference.

Other linkages between nucleotides and unnatural nucleotidescontemplated for the disclosed polynucleotides include those describedin U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265;5,658,873; 5,670,633; 5,792,747; and 5,700,920; U.S. Patent PublicationNo. 20040219565; International Patent Publication Nos. WO 98/39352 andWO 99/14226; Mesmaeker et. al., Current Opinion in Structural Biology5:343-355 (1995) and Susan M. Freier and Karl-Heinz Altmann, NucleicAcids Research, 25:4429-4443 (1997), the disclosures of which areincorporated herein by reference.

Specific examples of polynucleotides include those containing modifiedbackbones or non-natural internucleoside linkages. Polynucleotideshaving modified backbones include those that retain a phosphorus atom inthe backbone and those that do not have a phosphorus atom in thebackbone. Modified polynucleotides that do not have a phosphorus atom intheir internucleoside backbone are considered to be within the meaningof “polynucleotide.”

Modified polynucleotide backbones containing a phosphorus atom include,for example, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonates,5′-alkylene phosphonates and chiral phosphonates, phosphinates,phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Also contemplated are polynucleotides having inverted polaritycomprising a single 3′ to 3′ linkage at the 3′-most internucleotidelinkage, i.e. a single inverted nucleoside residue which may be abasic(the nucleotide is missing or has a hydroxyl group in place thereof).Salts, mixed salts and free acid forms are also contemplated.

Representative United States patents that teach the preparation of theabove phosphorus-containing linkages include, U.S. Pat. Nos. 3,687,808;4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423;5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939;5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821;5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599;5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, thedisclosures of which are incorporated by reference herein.

Modified polynucleotide backbones that do not include a phosphorus atomhave backbones that are formed by short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatom and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages; siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH2 component parts. In still otherembodiments, polynucleotides are provided with phosphorothioatebackbones and oligonucleosides with heteroatom backbones, and including—CH2-NH—O—CH2-, —CH2-N(CH3)-O—CH2-, —CH2-O—N(CH3)-CH2-,—CH2-N(CH3)-N(CH3)—CH2- and —O—N(CH3)-CH2-CH2- described in U.S. Pat.Nos. 5,489,677, and 5,602,240. See, for example, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, thedisclosures of which are incorporated herein by reference in theirentireties.

In various forms, the linkage between two successive monomers in thepolynucleotide consists of 2 to 4, desirably 3, groups/atoms selectedfrom —CH2-, —O—, —S—, —NRH—, >C═O, >C≡NRH, >C═S, —Si(R″)2-, —SO—,—S(O)2-, —P(O)2-, —PO(BH3)-, —P(O,S)—, —P(S)2-, —PO(R″)—, —PO(OCH3)-,and —PO(NHRH)—, where RH is selected from hydrogen and C1-4-alkyl, andR″ is selected from C1-6-alkyl and phenyl. Illustrative examples of suchlinkages are —CH2-CH2-CH2-, —CH2-CO—CH2-, —CH2-CHOH—CH2-, —O—CH2-O—,—O—CH2-CH2-, —O—CH2-CH=(including R5 when used as a linkage to asucceeding monomer), —CH2-CH2-O—, —NRH—CH2-CH2-, —CH2-CH2-NRH—,—CH2-NRH—CH2-, —O—CH2-CH2-NRH—, —NRH—CO—O—, —NRH—CO—NRH—, —NRH—CS—NRH—,—NRH—C(═NRH)—NRH—, —NRH—CO—CH2-NRH—O—CO—O—, —O—CO—CH2-O—, —O—CH2-CO—O—,—CH2-CO—NRH—, —O—CO—NRH—, —NRH—CO—CH2-, —O—CH2-CO—NRH—, —O—CH2-CH2-NRH—,—CH═N—O—, —CH2-NRH—O—, —CH2-O—N=(including R5 when used as a linkage toa succeeding monomer), —CH2-O—NRH—, —CO—NRH—CH2-, —CH2-NRH—O—,—CH2-NRH—CO—, —O—NRH—CH2-, —O—NRH, —O—CH2-S—, —S—CH2-O—, —CH2-CH2-S—,—O—CH2-CH2-S—, —S—CH2-CH=(including R5 when used as a linkage to asucceeding monomer), —S—CH2-CH2-, —S—CH2-CH2-O—, —S—CH2-CH2-S—,—CH2-S—CH2-, —CH2-SO—CH2-, —CH2-SO2-CH2-, —O—SO—O—, —O—S(O)2-O—,—O—S(O)2-CH2-, —O—S(O)2-NRH—, —NRH—S(O)2-CH2-; —O—S(O)2-CH2-,—O—P(O)2-O—, —O—P(O,S)—O—, —O—P(S)2-O—, —S—P(O)2-O—, —S—P(O,S)—O—,—S—P(S)2-O—, —O—P(O)2-S—, —O—P(O,S)—S—, —O—P(S)2-S—, —S—P(O)2-S—,—S—P(O,S)—S—, —S—P(S)2-S—, —O—PO(R″)—O—, —O—PO(OCH3)-O—, —O—PO(OCH2CH3)-O—, —O—PO(O CH2CH2S—R)—O—, —O—PO(BH3)-O—, —O—PO(NHRN)—O—,—O—P(O)2-NRH H—, —NRH—P(O)2-O—, —O—P(O,NRH)—O—, —CH2-P(O)2-O—,—O—P(O)2-CH2-, and —O—Si(R″)2-O—; among which —CH2-CO—NRH—, —CH2-NRH—O—,—S—CH2-O—, —O—P(O)2-O—O—P(—O,S)—O—, —O—P(S)2-O—, —NRH P(O)2-O—,—O—P(O,NRH)—O—, —O—PO(R″)—O—, —O—PO(CH3)-O—, and —O—PO(NHRN)—O—, whereRH is selected form hydrogen and C1-4-alkyl, and R″ is selected fromC1-6-alkyl and phenyl, are contemplated. Further illustrative examplesare given in Mesmaeker et. al., 1995, Current Opinion in StructuralBiology, 5: 343-355 and Susan M. Freier and Karl-Heinz Altmann, 1997,Nucleic Acids Research, vol 25: pp 4429-4443.

Still other modified forms of polynucleotides are described in detail inU.S. Patent Application No. 20040219565, the disclosure of which isincorporated by reference herein in its entirety.

Modified polynucleotides may also contain one or more substituted sugarmoieties. In certain aspects, polynucleotides comprise one of thefollowing at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkylor C2 to C10 alkenyl and alkynyl. Other embodiments includeO[(CH2)nO]mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, andO(CH2)nON[(CH2)nCH3]2, where n and m are from 1 to about 10. Otherpolynucleotides comprise one of the following at the 2′ position: C1 toC10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl,aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3,SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of a polynucleotide, or a group for improvingthe pharmacodynamic properties of a polynucleotide, and othersubstituents having similar properties. In one aspect, a modificationincludes 2′-methoxyethoxy (2′-O—CH2CH2OCH3, also known as2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., 1995, Hely. Chim. Acta,78: 486-504) i.e., an alkoxyalkoxy group. Other modifications include2′-dimethylaminooxyethoxy, i.e., a O(CH2)20N(CH3)2 group, also known as2′-DMAOE, and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-O—CH2-O—CH2-N(CH3)2.

Still other modifications include 2′-methoxy (2′-O—CH3), 2′-aminopropoxy(2′-OCH2CH2CH2NH2), 2′-allyl (2′-CH2-CH═CH2), 2′-O-allyl(2′-O—CH2-CH═CH2) and 2′-fluoro (2′-F). The 2′-modification may be inthe arabino (up) position or ribo (down) position. In one aspect, a2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the polynucleotide, for example, at the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedpolynucleotides and the 5′ position of 5′ terminal nucleotide.Polynucleotides may also have sugar mimetics such as cyclobutyl moietiesin place of the pentofuranosyl sugar. See, for example, U.S. Pat. Nos.4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137;5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722;5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873;5,670,633; 5,792,747; and 5,700,920, the disclosures of which areincorporated by reference in their entireties herein.

In one aspect, a modification of the sugar includes Locked Nucleic Acids(LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbonatom of the sugar ring, thereby forming a bicyclic sugar moiety. Thelinkage is in certain aspects a methylene (—CH2-)n group bridging the 2′oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs andpreparation thereof are described in WO 98/39352 and WO 99/14226, thedisclosures of which are incorporated herein by reference.

Polynucleotide Features

Each nanoconjugate provided comprises a plurality of biomolecules, andin various aspects, the biomolecules are polynucleotides. As a result,each nanoconjugate has the ability to bind to a plurality of targetpolynucleotides having a sufficiently complementary sequence. Forexample, if a specific polynucleotide is targeted, a singlenanoconjugate has the ability to bind to multiple copies of the samemolecule. In one aspect, methods are provided wherein the nanoconjugatecomprises identical polynucleotides, i.e., each polynucleotide has thesame length and the same sequence. In other aspects, the nanoconjugatecomprises two or more polynucleotides which are not identical, i.e., atleast one of the polynucleotides of the nanoconjugate differ from atleast one other polynucleotide of the nanoconjugate in that it has adifferent length and/or a different sequence. In aspects wherein ananoconjugate comprises different polynucleotides, these differentpolynucleotides bind to the same single target polynucleotide but atdifferent locations, or bind to different target polynucleotides whichencode different gene products. Accordingly, in various aspects, asingle nanoconjugate may be used in a method to inhibit expression ofmore than one gene product. Polynucleotides are thus used to targetspecific polynucleotides, whether at one or more specific regions in thetarget polynucleotide, or over the entire length of the targetpolynucleotide as the need may be to effect a desired level ofinhibition of gene expression.

Accordingly, in one aspect, the polynucleotides are designed withknowledge of the target sequence. Alternatively, a polynucleotide in ananoconjugate need not hybridize to a target biomolecule in order toachieve a desired effect as described herein. Regardless, methods ofmaking polynucleotides of a predetermined sequence are well-known. See,for example, Sambrook et al., Molecular Cloning: A Laboratory Manual(2nd ed. 1989) and F. Eckstein (ed.) Oligonucleotides and Analogues, 1stEd. (Oxford University Press, New York, 1991). Solid-phase synthesismethods are contemplated for both oligoribonucleotides andoligodeoxyribonucleotides (the well-known methods of synthesizing DNAare also useful for synthesizing RNA). Oligoribonucleotides andoligodeoxyribonucleotides can also be prepared enzymatically.

Alternatively, polynucleotides are selected from a library. Preparationof libraries of this type is well known in the art. See, for example,Oligonucleotide libraries: United States Patent Application 20050214782,published Sep. 29, 2005.

Polynucleotides contemplated for production of a nanoconjugate include,in one aspect, those which modulate expression of a gene productexpressed from a target polynucleotide. Accordingly, antisensepolynucleotides which hybridize to a target polynucleotide and inhibittranslation, siRNA polynucleotides which hybridize to a targetpolynucleotide and initiate an RNAse activity (for example RNAse H),triple helix forming polynucleotides which hybridize to double-strandedpolynucleotides and inhibit transcription, and ribozymes which hybridizeto a target polynucleotide and inhibit translation, are contemplated.

In some aspects, a polynucleotide-based nanoconjugate allows forefficient uptake of the nanoconjugate. In various aspects, thepolynucleotide comprises a nucleotide sequence that allows increaseduptake efficiency of the nanoconjugate. As used herein, “efficiency”refers to the number or rate of uptake of nanoconjugates in/by a cell.Because the process of nanoconjugates entering and exiting a cell is adynamic one, efficiency can be increased by taking up morenanoconjugates or by retaining those nanoconjugates that enter the cellfor a longer period of time. Similarly, efficiency can be decreased bytaking up fewer nanoconjugates or by retaining those nanoconjugates thatenter the cell for a shorter period of time.

Thus, the nucleotide sequence can be any nucleotide sequence that isdesired may be selected for, in various aspects, increasing ordecreasing cellular uptake of a nanoconjugate or gene regulation. Thenucleotide sequence, in some aspects, comprises a homopolymeric sequencewhich affects the efficiency with which the nanoparticle to which thepolynucleotide is attached is taken up by a cell. Accordingly, thehomopolymeric sequence increases or decreases the efficiency. It is alsocontemplated that, in various aspects, the nucleotide sequence is acombination of nucleobases, such that it is not strictly a homopolymericsequence. For example and without limitation, in various aspects, thenucleotide sequence comprises alternating thymidine and uridineresidues, two thymidines followed by two uridines or any combinationthat affects increased uptake is contemplated by the disclosure. In someaspects, the nucleotide sequence affecting uptake efficiency is includedas a domain in a polynucleotide comprising additional sequence. This“domain” would serve to function as the feature affecting uptakeefficiency, while the additional nucleotide sequence would serve tofunction, for example and without limitation, to regulate geneexpression. In various aspects, the domain in the polynucleotide can bein either a proximal, distal, or center location relative to thenanoconjugate. It is also contemplated that a polynucleotide comprisesmore than one domain.

The homopolymeric sequence, in some embodiments, increases theefficiency of uptake of the nanoconjugate by a cell. In some aspects,the homopolymeric sequence comprises a sequence of thymidine residues(polyT) or uridine residues (polyU). In further aspects, the polyT orpolyU sequence comprises two thymidines or uridines. In various aspects,the polyT or polyU sequence comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, about 55, about 60, about 65, about 70, about 75, about 80,about 85, about 90, about 95, about 100, about 125, about 150, about175, about 200, about 250, about 300, about 350, about 400, about 450,about 500 or more thymidine or uridine residues.

In some embodiments, it is contemplated that a nanoconjugate comprisinga polynucleotide that comprises a homopolymeric sequence is taken up bya cell with greater efficiency than a nanoconjugate comprising the samepolynucleotide but lacking the homopolymeric sequence. In variousaspects, a nanoconjugate comprising a polynucleotide that comprises ahomopolymeric sequence is taken up by a cell about 2-fold, about 3-fold,about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold,about 9-fold, about 10-fold, about 20-fold, about 30-fold, about40-fold, about 50-fold, about 100-fold or higher, more efficiently thana nanoconjugate comprising the same polynucleotide but lacking thehomopolymeric sequence.

In other aspects, the domain is a phosphate polymer (C3 residue). Insome aspects, the domain comprises a phosphate polymer (C3 residue) thatis comprised of two phosphates. In various aspects, the C3 residuecomprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, about 55, about 60,about 65, about 70, about 75, about 80, about 85, about 90, about 95,about 100, about 125, about 150, about 175, about 200, about 250, about300, about 350, about 400, about 450, about 500 or more phosphates.

In some embodiments, it is contemplated that a nanoconjugate comprisinga polynucleotide which comprises a domain is taken up by a cell withlower efficiency than a nanoconjugate comprising the same polynucleotidebut lacking the domain. In various aspects, a nanoconjugate comprising apolynucleotide which comprises a domain is taken up by a cell about2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about30-fold, about 40-fold, about 50-fold, about 100-fold or higher, lessefficiently than a nanoconjugate comprising the same polynucleotide butlacking the domain.

As used herein, a “conjugation site” is understood to mean a site on apolynucleotide to which a contrast agent is attached. In certainaspects, the disclosure also provides one or more polynucleotides thatare part of the nanoconjugate do not comprise a conjugation site whileone or more polynucleotides that are part of the same nanoconjugate docomprise a conjugation site. Conjugation of a contrast agent to ananoconjugate through a conjugation site is generally described inPCT/US2010/44844, which is incorporated herein by reference in itsentirety. The disclosure provides, in one aspect, a nanoconjugatecomprising a polynucleotide wherein the polynucleotide comprises one toabout ten conjugation sites. In another aspect, the polynucleotidecomprises five conjugation sites. In general, for a nucleotide, both itsbackbone (phosphate group) and nucleobase can be modified. Accordingly,the present disclosure contemplates that there are 2n conjugation sites,where n=length of the polynucleotide template. In related aspects, it iscontemplated that the composition comprises a nanoconjugate comprising aplurality of polynucleotides. In some aspects, the plurality ofpolynucleotides comprises at least one polynucleotide to which contrastagents are associated through one or more conjugation sites, as well asat least one polynucleotide that has gene regulatory activity asdescribed herein.

Accordingly, in some embodiments, it is contemplated that one or morepolynucleotides that are part of the nanoconjugate is not conjugated toa contrast agent while one or more polynucleotides that are part of thesame nanoconjugate are conjugated to a contrast agent.

The present disclosure also provides compositions comprising ananoconjugate, wherein the nanoconjugate comprises polynucleotides, andfurther comprising a transcriptional regulator, wherein thetranscriptional regulator induces transcription of a targetpolynucleotide in a target cell.

Polynucleotide Length

Nanoconjugates in the compositions and methods provided comprise, insome embodiments, a polynucleotide, or modified form thereof, which isfrom about 5 to about 100 nucleotides in length. Methods are alsocontemplated wherein the polynucleotide is about 5 to about 90nucleotides in length, about 5 to about 80 nucleotides in length, about5 to about 70 nucleotides in length, about 5 to about 60 nucleotides inlength, about 5 to about 50 nucleotides in length about 5 to about 45nucleotides in length, about 5 to about 40 nucleotides in length, about5 to about 35 nucleotides in length, about 5 to about 30 nucleotides inlength, about 5 to about 25 nucleotides in length, about 5 to about 20nucleotides in length, about 5 to about 15 nucleotides in length, about5 to about 10 nucleotides in length, and all polynucleotidesintermediate in length of the sizes specifically disclosed to the extentthat the polynucleotide is able to achieve the desired result.Accordingly, polynucleotides of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, and 100 or morenucleotides in length are contemplated.

Polynucleotide Copies—Same/Different Sequences

Nanoconjugates are provided which include those wherein a singlesequence in a single polynucleotide or multiple copies of the singlesequence in a single polynucleotide is part of a nanoconjugate. Thus, invarious aspects, a polynucleotide is contemplated with multiple copiesof a single sequence are in tandem, for example, two, three, four, five,six, seven eight, nine, ten or more tandem repeats.

Alternatively, the nanoconjugate includes at least two polynucleotideshaving different sequences. As above, the different polynucleotidesequences are in various aspects arranged in tandem (i.e., on a singlepolynucleotide) and/or in multiple copies (i.e., on at least twopolynucleotides). In methods wherein polynucleotides having differentsequences are part of the nanoconjugate, aspects of the disclosureinclude those wherein the different polynucleotide sequences hybridizeto different regions on the same polynucleotide. Alternatively, thedifferent polynucleotide sequences hybridize to differentpolynucleotides.

Polypeptides

As used herein a “polypeptide” refers to a polymer comprised of aminoacid residues. In some aspects of the disclosure, a nanoconjugatecomprises a polypeptide as described herein. Polypeptides are understoodin the art and include without limitation an antibody, an enzyme, astructural polypeptide and a hormone. In related aspects, thenanoconjugate comprising a polypeptide recognizes and associates with atarget molecule and enables detection of the target molecule. Apolypeptide of the disclosure can be a biomolecule or an additionalagent, each as described herein.

Polypeptides of the present disclosure may be either naturally occurringor non-naturally occurring. Polypeptides optionally include a spacer asdescribed herein above. As described above, a structural; polypeptidehas a crosslinking moiety through which the polypeptide is able tocrosslink with one or more other biomolecules in the nanoconjugatepreparative process. When a polypeptide is an additional agent, thepolypeptide need not, but may, include a crosslinking moiety. When theadditional polypeptide agent includes a crosslinking moiety, thepolypeptide generally does not crosslink to the nanoconjugate in amanner that is required for the nanoconjugate to maintain structuralintegrity.

Naturally Occurring Polypeptides

Naturally occurring polypeptides include without limitation biologicallyactive polypeptides (including antibodies) that exist in nature or canbe produced in a form that is found in nature by, for example, chemicalsynthesis or recombinant expression techniques. Naturally occurringpolypeptides also include lipoproteins and post-translationally modifiedproteins, such as, for example and without limitation, glycosylatedproteins.

Antibodies contemplated for use in the methods and compositions of thepresent disclosure include without limitation antibodies that recognizeand associate with a target molecule either in vivo or in vitro.

Structural polypeptides contemplated by the disclosure include withoutlimitation actin, tubulin, collagen, elastin, myosin, kinesin anddynein.

Non-Naturally Occurring Polypeptides

Non-naturally occurring polypeptides contemplated by the presentdisclosure include but are not limited to synthetic polypeptides, aswell as fragments, analogs and variants of naturally occurring ornon-naturally occurring polypeptides as defined herein. Non-naturallyoccurring polypeptides also include proteins or protein substances thathave D-amino acids, modified, derivatized, or non-naturally occurringamino acids in the D- or L-configuration and/or peptidomimetic units aspart of their structure. The term “protein” typically refers to largepolypeptides. The term “peptide” typically refers to short polypeptides.

Non-naturally occurring polypeptides are prepared, for example, using anautomated polypeptide synthesizer or, alternatively, using recombinantexpression techniques using a modified polynucleotide which encodes thedesired polypeptide.

As used herein a “fragment” of a polypeptide is meant to refer to anyportion of a polypeptide or protein smaller than the full-lengthpolypeptide or protein expression product.

As used herein an “analog” refers to any of two or more polypeptidessubstantially similar in structure and having the same biologicalactivity, but can have varying degrees of activity, to either the entiremolecule, or to a fragment thereof. Analogs differ in the composition oftheir amino acid sequences based on one or more mutations involvingsubstitution, deletion, insertion and/or addition of one or more aminoacids for other amino acids. Substitutions can be conservative ornon-conservative based on the physico-chemical or functional relatednessof the amino acid that is being replaced and the amino acid replacingit.

As used herein a “variant” refers to a polypeptide, protein or analogthereof that is modified to comprise additional chemical moieties notnormally a part of the molecule. Such moieties may modulate, for exampleand without limitation, the molecule's solubility, absorption, and/orbiological half-life. Moieties capable of mediating such effects aredisclosed in Remington's Pharmaceutical Sciences (1980). Procedures forcoupling such moieties to a molecule are well known in the art. Invarious aspects, polypeptides are modified by glycosylation, pegylation,and/or polysialylation.

Fusion proteins, including fusion proteins wherein one fusion componentis a fragment or a mimetic, are also contemplated. A “mimetic” as usedherein means a peptide or protein having a biological activity that iscomparable to the protein of which it is a mimetic. By way of example,an endothelial growth factor mimetic is a peptide or protein that has abiological activity comparable to the native endothelial growth factor.The term further includes peptides or proteins that indirectly mimic theactivity of a protein of interest, such as by potentiating the effectsof the natural ligand of the protein of interest.

This group of biomolecules includes antibodies along with fragments andderivatives thereof, including but not limited to Fab′ fragments, F(ab)2fragments, Fv fragments, Fc fragments, one or more complementaritydetermining regions (CDR) fragments, individual heavy chains, individuallight chain, dimeric heavy and light chains (as opposed toheterotetrameric heavy and light chains found in an intact antibody,single chain antibodies (scAb), humanized antibodies (as well asantibodies modified in the manner of humanized antibodies but with theresulting antibody more closely resembling an antibody in a non-humanspecies), chelating recombinant antibodies (CRABs), bispecificantibodies and multispecific antibodies, and other antibody derivativeor fragments known in the art.

Phospholipids

Also contemplated by the disclosure are nanoconjugates comprisingphospholipids. As discussed above for other biomolecules, a phospholipidbiomolecule includes, in certain aspects, an optional spacer component.A phospholipid that is a structural biomolecule includes a crosslinkingmoiety as described above for other biomolecules, through which thepohospholipid is able to crosslink to other biomolecules, A phospholipidthat is a non-structural biomolecule may, but need not, include acrosslinking moiety.

Lipid and phospholipid-derived hormones are contemplated as structuraland non-structural biomolecules, and these compounds derive from lipidssuch as linoleic acid and arachidonic acid and phospholipids. The mainclasses are the steroid hormones that derive from cholesterol and theeicosanoids.

In one specific embodiment, a synthetic high density lipoprotein (HDL)nanoconjugate is be constructed by modifying a gold nanoparticle with adense shell of phospholipids and APO1A. HDL is a dynamic serumnanoconjugate protective against the development of atherosclerosis andresultant illnesses such as heart disease and stroke. Likenaturally-occurring HDL, this synthetic construct is capable of bindingcholesterol in its hydrophobic phospholipid shell. It is the densepolyvalent arrangement of biological ligands on the surface of inorganicnanoparticles that imparts their unique ability to interact withbiological systems, regardless of their core material.

Metal Complexes

A metal complex as defined herein can be a structural non-biomoleculeand/or an additional agent, each as described herein. A metal complexoptional include a spacer as described herein above.

A “metal complex” as used herein refers to a metal and includes withoutlimitation a platinum compound as described herein, germanium(IV),titanium(IV), tin(IV), ruthenium(III), gold(III), and copper(II). If ametal complex is a structural non-biomolecule, it necessarily willinclude a crosslinking moiety through which is will crosslink to otherbiomolecules and/or non-biomolecules. A metal complex that is anadditional agent, may, but need not, include a crosslinking moiety.

Oligosaccharides

Oligosaccharides are contemplated by the disclosure to be a structuralbiomolecule and/or an additional agent, each as described herein.

Oligosaccharides include any carbohydrates comprising between about twoto about ten monosaccharides or more connected by either an alpha- orbeta-glycosidic link. Oligosaccharides are found throughout nature inboth the free and bound form. As discussed above for other biomolecules,a phospholipid biomolecule includes, in certain aspects, an optionalspacer component. An oligosaccharide that is a structural biomoleculeincludes a crosslinking moiety as described above for otherbiomolecules, through which the oligosaccharide is able to crosslink toother biomolecules, A oligosaccharide that is a non-structuralbiomolecule may, but need not, include a crosslinking moiety.Oligosaccharides optionally include a spacer as described herein above.

Other Non-Biomolecules

A non-biomolecule as used herein is selected from the group consistingof a diluent molecule, a metal complex as described above and anynon-carbon containing molecule known in the art.

Nanoconjugate Structure

As described herein, the structure of each nanoconjugate is defined by(i) the surface that was used in the manufacture of the nanoconjugates(ii) the type of biomolecules forming the nanoconjugate, and (iii) thedegree and type of crosslinking between individual biomolecules onand/or around the surface. Also as discussed herein, in every aspect ofthe nanoconjugate provided, the biomolecules, with or without anon-biomolecule, are crosslinked. The crosslinking is effected throughthe use of one or more crosslinking moieties.

Crosslinking

Crosslinking moieties contemplated by the disclosure include but are notlimited to an amine, amide, alcohol, ester, aldehyde, ketone, thiol,disulfide, carboxylic acid, phenol, imidazole, hydrazine, hydrazone,azide and an alkyne. Any crosslinking moiety can be used, so long as itcan be attached to a biomolecule and/or non-biomolecule by a methodknown to one of skill in the art.

In various embodiments, an alkyne is associated with a biomoleculethrough a degradable moiety. For example and without limitation, thealkyne in various aspects is associated with a biomolecule through anacid-labile moiety that is degraded upon entry into an endosome inside acell.

In some aspects, the surface with which a biomolecule is associated actsas a catalyst for the crosslinking moieties. Under appropriateconditions, contact of a crosslinking moiety with the surface willactivate the crosslinking moiety, thereby initiating sometimesspontaneous crosslinking between structural biomolecules and/ornon-biomolecules. In one specific aspect, the crosslinking moiety is analkyne and the surface is comprised of gold. In this aspect, and asdescribed herein, the gold surface acts as a catalyst to activate analkyne crosslinking moiety, thus allowing the crosslink to form betweena biomolecule comprising an alkyne crosslinking moiety to anotherbiomolecule comprising an alkyne crosslinking moiety.

Production methods are also contemplated wherein a chemical is used toeffect the crosslinking between biomolecules. Chemicals contemplated foruse in crosslinking biomolecules are discussed below.

Polynucleotides contemplated for use in the methods include thoseassociated with a nanoconjugate through any means. Regardless of themeans by which the polynucleotide is associated with the nanoconjugate,association in various aspects is effected through a 5′ linkage, a 3′linkage, some type of internal linkage, or any combination of theseattachments and depends on the location of the crosslinking moiety inthe polynucleotide. By way of example, a crosslinking moiety on the 3′end of a polynucleotide means that the polynucleotide will associatewith the nanoconjugate at its 3′ end.

In various aspects, the crosslinking moiety is located in a spacer. Aspacer is described herein above, and it is contemplated that anucleotide in the spacer comprises a crosslinking moiety. In furtheraspects, a nucleotide in the spacer comprises more than one crosslinkingmoiety, and the more than one crosslinking moieties are either the sameor different. In addition, each nucleotide in a spacer can comprise oneor more crosslinking moieties, which can either be the same ordifferent.

In some embodiments, the polynucleotide does not comprise a spacer. Inthese aspects, the polynucleotide comprises one or more crosslinkingmoieties along its length. The crosslinking moieties can be the same ordifferent, and each nucleotide in the polynucleotide can comprise one ormore crosslinking moieties, and these too can either be the same ordifferent.

In one aspect, a polynucleotide comprises one crosslinking moiety, whichcan optionally be in a spacer portion of the polynucleotide if a spaceris present. If a polynucleotide consists of one crosslinking moiety,then it is contemplated that the polynucleotide is an additional agentthat is crosslinked to a nanoconjugate.

In further aspects, a polynucleotide comprises from about 1 to about 500crosslinking moieties, or from about 1 to about 100, or from about 5 toabout 50, or from about 10 to about 30, or from about 10 to about 20crosslinking moieties. In various embodiments, the polynucleotidecomprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60 or more crosslinking moieties. Inaspects wherein the spacer comprises more than one crosslinking moiety,the moieties can all be the same or they can be different, and anycombination of crosslinking moieties may be used.

In one aspect, the crosslinking moiety is located in the same positionin each polynucleotide, which under certain conditions orients all ofthe polynucleotides in the same direction. In some aspects, thedirection is such that the 5′ and 3′ ends of a polynucleotide arediametrically opposed to each other. In these aspects, the spacer endwill be more “proximal” with respect to the nanoconjugate surface, whilethe opposite end will be more “distal” with respect to the nanoconjugatesurface. With respect to “proximal” and “distal” and their relationshipto the nanoconjugate surface, it will be understood that the location isdetermined when the surface is present, and prior to its optional atleast partial removal. The orienting of polynucleotides in the samedirection in a nanoconjugate is useful, for example and withoutlimitation, when a polynucleotide is to be hybridized to a targetbiomolecule since the nanoconjugate structure provides a polyvalentnetwork of polynucleotides that are positioned to recognize andassociate with the target biomolecule.

In another aspect, the crosslinking moiety is located in differentpositions in the polynucleotides, which under certain conditions canprovide mixed orientation of the polynucleotides after crosslinking.

In some embodiments, a biomolecule and/or non-biomolecule comprising acrosslinking moiety is attached to a nanoparticle, wherein theattachment is displaceable. Thus, in one aspect, a crosslinking moietythat associates with a surface can remain in association with thesurface, or it can be displaced from the surface through reaction withanother crosslinking moiety that is present on another biomoleculeand/or non-biomolecule. As previously described, in some embodiments theassociation of a biomolecule and/or non-biomolecule comprising acrosslinking moiety with a surface results in the crosslinking of thebiomolecule and/or non-biomolecule to another biomolecule and/ornon-biomolecule that is in association with the surface.

Nanoparticles Providing Shape

The shape of each nanoconjugate in the plurality is determined in partby the surface used in its production, and in part by the biomoleculesand/or non-biomolecules used in its production. The surface is invarious aspects planar or three dimensional. Thus, in various aspects,the surface is a nanoparticle.

In general, nanoparticles contemplated include any compound or substancewith a high loading capacity for a biomolecule to effect the productionof a nanoconjugate as described herein, including for example andwithout limitation, a metal, a semiconductor, and an insulator particlecompositions, and a dendrimer (organic versus inorganic).

Thus, nanoparticles are contemplated which comprise a variety ofinorganic materials including, but not limited to, metals,semi-conductor materials or ceramics as described in US patentapplication No 20030147966. For example, metal-based nanoparticlesinclude those described herein. Ceramic nanoparticle materials include,but are not limited to, brushite, tricalcium phosphate, alumina, silica,and zirconia. Organic materials from which nanoparticles are producedinclude carbon. Nanoparticle polymers include polystyrene, siliconerubber, polycarbonate, polyurethanes, polypropylenes,polymethylmethacrylate, polyvinyl chloride, polyesters, polyethers, andpolyethylene. Biodegradable, biopolymer (e.g. polypeptides such as BSA,polysaccharides, etc.), other biological materials (e.g. carbohydrates),and/or polymeric compounds are also contemplated for use in producingnanoparticles.

In one embodiment, the nanoparticle is metallic, and in various aspects,the nanoparticle is a colloidal metal. Thus, in various embodiments,nanoparticles useful in the practice of the methods include metal(including for example and without limitation, gold, silver, platinum,aluminum, palladium, copper, cobalt, iron, indium, nickel, or any othermetal amenable to nanoparticle formation), semiconductor (including forexample and without limitation, CdSe, CdS, and CdS or CdSe coated withZnS) and magnetic (for example, ferromagnetite) colloidal materials.Other nanoparticles useful in the practice of the invention include,also without limitation, ZnS, ZnO, Ti, TiO2, Sn, SnO2, Si, SiO2, Fe,Fe+4, Fe3O4, Fe2O3, Ag, Cu, Ni, Al, steel, cobalt-chrome alloys, Cd,titanium alloys, AgI, AgBr, HgI2, PbS, PbSe, ZnTe, CdTe, In2S3, In2Se3,Cd3P2, Cd3As2, InAs, and GaAs. Methods of making ZnS, ZnO, TiO2, AgI,AgBr, HgI2, PbS, PbSe, ZnTe, CdTe, In2S3, In2Se3, Cd3P2, Cd3As2, InAs,and GaAs nanoparticles are also known in the art. See, e.g., Weller,Angew. Chem. Int. Ed. Engl., 32, 41 (1993); Henglein, Top. Curr. Chem.,143, 113 (1988); Henglein, Chem. Rev., 89, 1861 (1989); Brus, Appl.Phys. A., 53, 465 (1991); Bahncmann, in Photochemical Conversion andStorage of Solar Energy (eds. Pelizetti and Schiavello 1991), page 251;Wang and Herron, J. Phys. Chem., 95, 525 (1991); Olshaysky, et al., J.Am. Chem. Soc., 112, 9438 (1990); Ushida et al., J. Phys. Chem., 95,5382 (1992).

In practice, compositions and methods are provided using any suitablenanoparticle suitable for use in methods to the extent they do notinterfere with complex formation. The size, shape and chemicalcomposition of the particles contribute to the properties of theresulting nanoconjugate. These properties include for example, opticalproperties, optoelectronic properties, electrochemical properties,electronic properties, stability in various solutions, magneticproperties, and pore and channel size variation. The use of mixtures ofparticles having different sizes, shapes and/or chemical compositions,as well as the use of nanoparticles having uniform sizes, shapes andchemical composition, is contemplated. Examples of suitable particlesinclude, without limitation, nanoparticles, aggregate particles,isotropic (such as spherical particles) and anisotropic particles (suchas non-spherical rods, tetrahedral, prisms) and core-shell particlessuch as the ones described in U.S. patent application Ser. No.10/034,451, filed Dec. 28, 2002 and International application no.PCT/US01/50825, filed Dec. 28, 2002, the disclosures of which areincorporated by reference in their entirety.

Methods of making metal, semiconductor and magnetic nanoparticles arewell-known in the art. See, for example, Schmid, G. (ed.) Clusters andColloids (V C H, Weinheim, 1994); Hayat, M. A. (ed.) Colloidal Gold:Principles, Methods, and Applications (Academic Press, San Diego, 1991);Massart, R., IEEE Transactions On Magnetics, 17, 1247 (1981); Ahmadi, T.S. et al., Science, 272, 1924 (1996); Henglein, A. et al., J. Phys.Chem., 99, 14129 (1995); Curtis, A. C., et al., Angew. Chem. Int. Ed.Engl., 27, 1530 (1988). Preparation of polyalkylcyanoacrylatenanoparticles prepared is described in Fattal, et al., J. ControlledRelease (1998) 53: 137-143 and U.S. Pat. No. 4,489,055. Methods formaking nanoparticles comprising poly(D-glucaramidoamine)s are describedin Liu, et al., J. Am. Chem. Soc. (2004) 126:7422-7423. Preparation ofnanoparticles comprising polymerized methylmethacrylate (MMA) isdescribed in Tondelli, et al., Nucl. Acids Res. (1998) 26:5425-5431, andpreparation of dendrimer nanoparticles is described in, for exampleKukowska-Latallo, et al., Proc. Natl. Acad. Sci. USA (1996) 93:4897-4902(Starburst polyamidoamine dendrimers)

Also as described in US patent application No 20030147966, nanoparticlescomprising materials described herein are available commercially from,for example, Ted Pella, Inc. (gold), Amersham Corporation (gold) andNanoprobes, Inc. (gold), or they can be produced from progressivenucleation in solution (e.g., by colloid reaction), or by variousphysical and chemical vapor deposition processes, such as sputterdeposition. See, e.g., HaVashi, (1987) Vac. Sci. Technol. July/August1987, A5(4):1375-84; Hayashi, (1987) Physics Today, December 1987, pp.44-60; MRS Bulletin, January 1990, pgs. 16-47.

As further described in US patent application No 20030147966,nanoparticles contemplated are produced using HAuC14 and acitrate-reducing agent, using methods known in the art. See, e.g.,Marinakos et al., (1999) Adv. Mater. 11: 34-37; Marinakos et al., (1998)Chem. Mater. 10: 1214-19; Enustun & Turkevich, (1963) J. Am. Chem. Soc.85: 3317. Tin oxide nanoparticles having a dispersed aggregate particlesize of about 140 nm are available commercially from VacuumMetallurgical Co., Ltd. of Chiba, Japan. Other commercially availablenanoparticles of various compositions and size ranges are available, forexample, from Vector Laboratories, Inc. of Burlingame, Calif.

Nanoparticle Size

In various aspects, methods provided include those utilizingnanoparticles which range in size from about 1 nm to about 250 nm inmean diameter, about 1 nm to about 240 nm in mean diameter, about 1 nmto about 230 nm in mean diameter, about 1 nm to about 220 nm in meandiameter, about 1 nm to about 210 nm in mean diameter, about 1 nm toabout 200 nm in mean diameter, about 1 nm to about 190 nm in meandiameter, about 1 nm to about 180 nm in mean diameter, about 1 nm toabout 170 nm in mean diameter, about 1 nm to about 160 nm in meandiameter, about 1 nm to about 150 nm in mean diameter, about 1 nm toabout 140 nm in mean diameter, about 1 nm to about 130 nm in meandiameter, about 1 nm to about 120 nm in mean diameter, about 1 nm toabout 110 nm in mean diameter, about 1 nm to about 100 nm in meandiameter, about 1 nm to about 90 nm in mean diameter, about 1 nm toabout 80 nm in mean diameter, about 1 nm to about 70 nm in meandiameter, about 1 nm to about 60 nm in mean diameter, about 1 nm toabout 50 nm in mean diameter, about 1 nm to about 40 nm in meandiameter, about 1 nm to about 30 nm in mean diameter, or about 1 nm toabout 20 nm in mean diameter, about 1 nm to about 10 nm in meandiameter. In other aspects, the size of the nanoparticles is from about5 nm to about 150 nm (mean diameter), from about 5 to about 50 nm, fromabout 10 to about 30 nm. The size of the nanoparticles is from about 5nm to about 150 nm (mean diameter), from about 30 to about 100 nm, fromabout 40 to about 80 nm. The size of the nanoparticles used in a methodvaries as required by their particular use or application. The variationof size is advantageously used to optimize certain physicalcharacteristics of the nanoparticles, for example, optical properties oramount surface area that can be derivatized as described herein.

Biomolecule Density

Nanoconjugates as provided herein have a density of the biomolecules onthe surface of the nanoconjugate that is, in various aspects, sufficientto result in cooperative behavior between nanoconjugates and betweenbiomolecules on a single nanoconjugate. In another aspect, thecooperative behavior between the nanoconjugates increases the resistanceof the biomolecule to degradation, and provides a sharp meltingtransition relative to biomolecules that are not part of ananoconjugate. In one aspect, the uptake of nanoconjugates by a cell isinfluenced by the density of polynucleotides associated with thenanoparticle. As described in PCT/US2008/65366, incorporated herein byreference in its entirety, a higher density of polynucleotides on thesurface of a polynucleotide functionalized nanoparticle is associatedwith an increased uptake of nanoparticles by a cell. This aspect islikewise contemplated to be a property of nanoconjugates, wherein ahigher density of biomolecules that make up a nanoconjugate isassociated with an increased uptake of a nanoconjugate by a cell.

A surface density adequate to make the nanoconjugates stable and theconditions necessary to obtain it for a desired combination ofnanoconjugates and biomolecules can be determined empirically. Broadly,the smaller the biomolecule and/or non-biomolecule that is used, thehigher the surface density of that biomolecule and/or non-biomoleculecan be. Generally, a surface density of at least 2 pmol/cm² will beadequate to provide stable nanoconjugate-compositions. In some aspects,the surface density is at least 15 pmol/cm². Methods are also providedwherein the biomolecule is present in a nanoconjugate at a surfacedensity of at least 2 pmol/cm2, at least 3 pmol/cm2, at least 4pmol/cm2, at least 5 pmol/cm2, at least 6 pmol/cm2, at least 7 pmol/cm2,at least 8 pmol/cm2, at least 9 pmol/cm2, at least 10 pmol/cm2, at leastabout 15 pmol/cm2, at least about 20 pmol/cm2, at least about 25pmol/cm2, at least about 30 pmol/cm2, at least about 35 pmol/cm2, atleast about 40 pmol/cm2, at least about 45 pmol/cm2, at least about 50pmol/cm2, at least about 55 pmol/cm2, at least about 60 pmol/cm2, atleast about 65 pmol/cm2, at least about 70 pmol/cm2, at least about 75pmol/cm2, at least about 80 pmol/cm2, at least about 85 pmol/cm2, atleast about 90 pmol/cm2, at least about 95 pmol/cm2, at least about 100pmol/cm2, at least about 125 pmol/cm2, at least about 150 pmol/cm2, atleast about 175 pmol/cm2, at least about 200 pmol/cm2, at least about250 pmol/cm2, at least about 300 pmol/cm2, at least about 350 pmol/cm2,at least about 400 pmol/cm2, at least about 450 pmol/cm2, at least about500 pmol/cm2, at least about 550 pmol/cm2, at least about 600 pmol/cm2,at least about 650 pmol/cm2, at least about 700 pmol/cm2, at least about750 pmol/cm2, at least about 800 pmol/cm2, at least about 850 pmol/cm2,at least about 900 pmol/cm2, at least about 950 pmol/cm2, at least about1000 pmol/cm2 or more.

It is contemplated that the density of polynucleotides in ananoconjugate modulates specific biomolecule and/or non-biomoleculeinteractions with the polynucleotide on the surface and/or with thenanoconjugate itself. Under various conditions, some polypeptides may beprohibited from interacting with polynucleotides that are part of ananoconjugate based on steric hindrance caused by the density ofpolynucleotides. In aspects where interaction of polynucleotides with abiomolecule and/or non-biomolecule that are otherwise precluded bysteric hindrance is desirable, the density of polynucleotides in thenanoconjugate is decreased to allow the biomolecule and/ornon-biomolecule to interact with the polynucleotide.

Nanoparticles of larger diameter are, in some aspects, contemplated tobe templated with a greater number of polynucleotides [Hurst et al.,Analytical Chemistry 78(24): 8313-8318 (2006)] during nanoconjugateproduction. In some aspects, therefore, the number of polynucleotidesused in the production of a nanoconjugate is from about 10 to about25,000 polynucleotides per nanoconjugate. In further aspects, the numberof polynucleotides used in the production of a nanoconjugate is fromabout 50 to about 10,000 polynucleotides per nanoconjugate, and in stillfurther aspects the number of polynucleotides used in the production ofa nanoconjugate is from about 200 to about 5,000 polynucleotides pernanoconjugate. In various aspects, the number of polynucleotides used inthe production of a nanoconjugate is about 10, about 15, about 20, about25, about 30, about 35, about 40, about 45, about 50, about 55, about60, about 65, about 70, about 75, about 80, about 85, about 90, about95, about 100, about 105, about 110, about 115, about 120, about 125,about 130, about 135, about 140, about 145, about 150, about 155, about160, about 165, about 170, about 175, about 180, about 185, about 190,about 195, about 200, about 205, about 210, about 215, about 220, about225, about 230, about 235, about 240, about 245, about 250, about 255,about 260, about 265, about 270, about 275, about 280, about 285, about290, about 295, about 300, about 305, about 310, about 315, about 320,about 325, about 330, about 335, about 340, about 345, about 350, about355, about 360, about 365, about 370, about 375, about 380, about 385,about 390, about 395, about 400, about 405, about 410, about 415, about420, about 425, about 430, about 435, about 440, about 445, about 450,about 455, about 460, about 465, about 470, about 475, about 480, about485, about 490, about 495, about 500, about 505, about 510, about 515,about 520, about 525, about 530, about 535, about 540, about 545, about550, about 555, about 560, about 565, about 570, about 575, about 580,about 585, about 590, about 595, about 600, about 605, about 610, about615, about 620, about 625, about 630, about 635, about 640, about 645,about 650, about 655, about 660, about 665, about 670, about 675, about680, about 685, about 690, about 695, about 700, about 705, about 710,about 715, about 720, about 725, about 730, about 735, about 740, about745, about 750, about 755, about 760, about 765, about 770, about 775,about 780, about 785, about 790, about 795, about 800, about 805, about810, about 815, about 820, about 825, about 830, about 835, about 840,about 845, about 850, about 855, about 860, about 865, about 870, about875, about 880, about 885, about 890, about 895, about 900, about 905,about 910, about 915, about 920, about 925, about 930, about 935, about940, about 945, about 950, about 955, about 960, about 965, about 970,about 975, about 980, about 985, about 990, about 995, about 1000, about1100, about 1200, about 1300, about 1400, about 1500, about 1600, about1700, about 1800, about 1900, about 2000, about 2100, about 2200, about2300, about 2400, about 2500, about 2600, about 2700, about 2800, about2900, about 3000, about 3100, about 3200, about 3300, about 3400, about3500, about 3600, about 3700, about 3800, about 3900, about 4000, about4100, about 4200, about 4300, about 4400, about 4500, about 4600, about4700, about 4800, about 4900, about 5000, about 5100, about 5200, about5300, about 5400, about 5500, about 5600, about 5700, about 5800, about5900, about 6000, about 6100, about 6200, about 6300, about 6400, about6500, about 6600, about 6700, about 6800, about 6900, about 7000, about7100, about 7200, about 7300, about 7400, about 7500, about 7600, about7700, about 7800, about 7900, about 8000, about 8100, about 8200, about8300, about 8400, about 8500, about 8600, about 8700, about 8800, about8900, about 9000, about 9100, about 9200, about 9300, about 9400, about9500, about 9600, about 9700, about 9800, about 9900, about 10000, about10100, about 10200, about 10300, about 10400, about 10500, about 10600,about 10700, about 10800, about 10900, about 11000, about 11100, about11200, about 11300, about 11400, about 11500, about 11600, about 11700,about 11800, about 11900, about 12000, about 12100, about 12200, about12300, about 12400, about 12500, about 12600, about 12700, about 12800,about 12900, about 13000, about 13100, about 13200, about 13300, about13400, about 13500, about 13600, about 13700, about 13800, about 13900,about 14000, about 14100, about 14200, about 14300, about 14400, about14500, about 14600, about 14700, about 14800, about 14900, about 15000,about 15100, about 15200, about 15300, about 15400, about 15500, about15600, about 15700, about 15800, about 15900, about 16000, about 16100,about 16200, about 16300, about 16400, about 16500, about 16600, about16700, about 16800, about 16900, about 17000, about 17100, about 17200,about 17300, about 17400, about 17500, about 17600, about 17700, about17800, about 17900, about 18000, about 18100, about 18200, about 18300,about 18400, about 18500, about 18600, about 18700, about 18800, about18900, about 19000, about 19100, about 19200, about 19300, about 19400,about 19500, about 19600, about 19700, about 19800, about 19900, about20000, about 20100, about 20200, about 20300, about 20400, about 20500,about 20600, about 20700, about 20800, about 20900, about 21000, about21100, about 21200, about 21300, about 21400, about 21500, about 21600,about 21700, about 21800, about 21900, about 22000, about 22100, about22200, about 22300, about 22400, about 22500, about 22600, about 22700,about 22800, about 22900, about 23000, about 23100, about 23200, about23300, about 23400, about 23500, about 23600, about 23700, about 23800,about 23900, about 24000, about 24100, about 24200, about 24300, about24400, about 24500, about 24600, about 24700, about 24800, about 24900,about 25000 or more per nanoconjugate.

It is also contemplated that polynucleotide surface density modulatesthe stability of the polynucleotide associated with the nanoconjugate.Thus, in one embodiment, a nanoconjugate comprising a polynucleotide isprovided wherein the polynucleotide has a half-life that is at leastsubstantially the same as the half-life of an identical polynucleotidethat is not part of a nanoconjugate. In other embodiments, thepolynucleotide associated with the nanoparticle has a half-life that isabout 5% greater to about 1,000,000-fold greater or more than thehalf-life of an identical polynucleotide that is not part of ananoconjugate.

Hollow Nanoconjugates

As described herein, in various aspects the nanoconjugates provided bythe disclosure are hollow. The porosity and/or rigidity of a hollownanoconjugate depends in part on the density of biomolecules, andnon-biomolecules when present, that are crosslinked on the surface of ananoparticle during nanoconjugate production. In general, a lowerdensity of biomolecules crosslinked on the surface of the nanoparticleresults in a more porous nanoconjugate, while a higher density ofbiomolecules crosslinked on the surface of the nanoparticle results in amore rigid nanoconjugate. Porosity and density of a hollow nanoconjugatealso depends on the degree and type of crosslinking between biomoleculesand/or non-biomolecules.

In some aspects, a hollow nanoconjugate is produced which is then loadedwith a desirable additional agent, and the nanoconjugate is then coveredwith a coating to prevent the escape of the additional agent. Thecoating, in some aspects, is also an additional agent and is describedin more detail below.

Additional Agents

Additional agents contemplated by the disclosure include a biomolecule,non-biomolecule, detectable marker, a coating, a polymeric agent, acontrast agent, an embolic agent, a short internal complementarypolynucleotide (sicPN), a transcriptional regulator, a therapeuticagent, an antibiotic and a targeting moiety.

Therapeutic Agents

“Therapeutic agent,” “drug” or “active agent” as used herein means anycompound useful for therapeutic or diagnostic purposes. The terms asused herein are understood to mean any compound that is administered toa patient for the treatment of a condition that can traverse a cellmembrane more efficiently when attached to a nanoparticle ornanoconjugate of the disclosure than when administered in the absence ofa nanoparticle or nanoconjugate of the disclosure.

The present disclosure is applicable to any therapeutic agent for whichdelivery is desired. Non-limiting examples of such active agents as wellas hydrophobic drugs are found in U.S. Pat. No. 7,611,728, which isincorporated by reference herein in its entirety.

Compositions and methods disclosed herein, in various embodiments, areprovided wherein the nanoconjugate comprises a multiplicity oftherapeutic agents. In one aspect, compositions and methods are providedwherein the multiplicity of therapeutic agents are specifically attachedto one nanoconjugate. In another aspect, the multiplicity of therapeuticagents is specifically attached to more than one nanoconjugate.

Therapeutic agents useful in the materials and methods of the presentdisclosure can be determined by one of ordinary skill in the art. Forexample and without limitation, and as exemplified herein, one canperform a routine in vitro test to determine whether a therapeutic agentis able to traverse the cell membrane of a cell more effectively whenattached to a nanoconjugate than in the absence of attachment to thenanoconjugate.

In various embodiments, a drug delivery composition is providedcomprising a nanoconjugate and a therapeutic agent, the therapeuticagent being one that is deliverable at a significantly lower level inthe absence of attachment of the therapeutic agent to the nanogonjugatecompared to the delivery of the therapeutic agent when attached to thenanogonjugate, and wherein the ratio of polynucleotide on thenanogonjugate to the therapeutic agent attached to the nanogonjugate issufficient to allow transport of the therapeutic agent into a cell. Asused herein, “ratio” refers to a number comparison of polynucleotide totherapeutic agent. For example and without limitation, a 1:1 ratiorefers to there being one polynucleotide molecule for every therapeuticagent molecule that is attached to a nanogonjugate.

In one embodiment, methods and compositions are provided wherein atherapeutic agent is able to traverse a cell membrane more efficientlywhen attached to a nanoconjugate than when it is not attached to thenanoconjugate. In various aspects, a therapeutic agent is able totraverse a cell membrane about 2-fold, about 3-fold, about 4-fold, about5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about10-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold,about 60-fold, about 70-fold, about 80-fold, about 90-fold or about100-fold or more efficiently when attached to a nanoconjugate than whenit is not attached to the nanoconjugate.

Therapeutic agents include but are not limited to hydrophilic andhydrophobic compounds. Accordingly, therapeutic agents contemplated bythe present disclosure include without limitation drug-like molecules,biomolecules and non-biomolecules.

Protein therapeutic agents include, without limitation peptides,enzymes, structural proteins, receptors and other cellular orcirculating proteins as well as fragments and derivatives thereof, theaberrant expression of which gives rise to one or more disorders.Therapeutic agents also include, as one specific embodiment,chemotherapeutic agents. Therapeutic agents also include, in variousembodiments, a radioactive material.

In various aspects, protein therapeutic agents include cytokines orhematopoietic factors including without limitation IL-1 alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-11, colony stimulating factor-1(CSF-1), M-CSF, SCF, GM-CSF, granulocyte colony stimulating factor(G-CSF), interferon-alpha (IFN-alpha), consensus interferon, IFN-beta,IFN-gamma, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16,IL-17, IL-18, erythropoietin (EPO), thrombopoietin (TPO), angiopoietins,for example Ang-1, Ang-2, Ang-4, Ang-Y, the human angiopoietin-likepolypeptide, vascular endothelial growth factor (VEGF), angiogenin, bonemorphogenic protein-1, bone morphogenic protein-2, bone morphogenicprotein-3, bone morphogenic protein-4, bone morphogenic protein-5, bonemorphogenic protein-6, bone morphogenic protein-7, bone morphogenicprotein-8, bone morphogenic protein-9, bone morphogenic protein-10, bonemorphogenic protein-11, bone morphogenic protein-12, bone morphogenicprotein-13, bone morphogenic protein-14, bone morphogenic protein-15,bone morphogenic protein receptor IA, bone morphogenic protein receptorIB, brain derived neurotrophic factor, ciliary neutrophic factor,ciliary neutrophic factor receptor, cytokine-induced neutrophilchemotactic factor 1, cytokine-induced neutrophil, chemotactic factor2α, cytokine-induced neutrophil chemotactic factor 2β, β endothelialcell growth factor, endothelin 1, epidermal growth factor,epithelial-derived neutrophil attractant, fibroblast growth factor 4,fibroblast growth factor 5, fibroblast growth factor 6, fibroblastgrowth factor 7, fibroblast growth factor 8, fibroblast growth factor8b, fibroblast growth factor 8c, fibroblast growth factor 9, fibroblastgrowth factor 10, fibroblast growth factor acidic, fibroblast growthfactor basic, glial cell line-derived neutrophic factor receptor α1,glial cell line-derived neutrophic factor receptor α2, growth relatedprotein, growth related protein a, growth related protein β, growthrelated protein γ, heparin binding epidermal growth factor, hepatocytegrowth factor, hepatocyte growth factor receptor, insulin-like growthfactor I, insulin-like growth factor receptor, insulin-like growthfactor II, insulin-like growth factor binding protein, keratinocytegrowth factor, leukemia inhibitory factor, leukemia inhibitory factorreceptor α, nerve growth factor nerve growth factor receptor,neurotrophin-3, neurotrophin-4, placenta growth factor, placenta growthfactor 2, platelet-derived endothelial cell growth factor, plateletderived growth factor, platelet derived growth factor A chain, plateletderived growth factor AA, platelet derived growth factor AB, plateletderived growth factor B chain, platelet derived growth factor BB,platelet derived growth factor receptor α, platelet derived growthfactor receptor β, pre-B cell growth stimulating factor, stem cellfactor receptor, TNF, including TNF0, TNF1, TNF2, transforming growthfactor α, transforming growth factor β, transforming growth factor β1,transforming growth factor β1.2, transforming growth factor β2,transforming growth factor β3, transforming growth factor β5, latenttransforming growth factor β1, transforming growth factor β bindingprotein I, transforming growth factor β binding protein II, transforminggrowth factor β binding protein III, tumor necrosis factor receptor typeI, tumor necrosis factor receptor type II, urokinase-type plasminogenactivator receptor, vascular endothelial growth factor, and chimericproteins and biologically or immunologically active fragments thereof.Examples of biologic agents include, but are not limited to,immuno-modulating proteins such as cytokines, monoclonal antibodiesagainst tumor antigens, tumor suppressor genes, and cancer vaccines.Examples of interleukins that may be used in conjunction with thecompositions and methods of the present invention include, but are notlimited to, interleukin 2 (IL-2), and interleukin 4 (IL-4), interleukin12 (IL-12). Other immuno-modulating agents other than cytokines include,but are not limited to bacillus Calmette-Guerin, levamisole, andoctreotide.

As described by the present disclosure, in some aspects therapeuticagents include small molecules. The term “small molecule,” as usedherein, refers to a chemical compound, for instance a peptidometic thatmay optionally be derivatized, or any other low molecular weight organiccompound, either natural or synthetic. Such small molecules may be atherapeutically deliverable substance or may be further derivatized tofacilitate delivery.

By “low molecular weight” is meant compounds having a molecular weightof less than 1000 Daltons, typically between 300 and 700 Daltons. Lowmolecular weight compounds, in various aspects, are about 100, about150, about 200, about 250, about 300, about 350, about 400, about 450,about 500, about 550, about 600, about 650, about 700, about 750, about800, about 850, about 900, about 1000 or more Daltons.

The term “drug-like molecule” is well known to those skilled in the art,and includes the meaning of a compound that has characteristics thatmake it suitable for use in medicine, for example and without limitationas the active agent in a medicament. Thus, for example and withoutlimitation, a drug-like molecule is a molecule that is synthesized bythe techniques of organic chemistry, or by techniques of molecularbiology or biochemistry, and is in some aspects a small molecule asdefined herein. A drug-like molecule, in various aspects, additionallyexhibits features of selective interaction with a particular protein orproteins and is bioavailable and/or able to penetrate cellular membraneseither alone or in combination with a composition or method of thepresent disclosure.

In various embodiments, therapeutic agents described in U.S. Pat. No.7,667,004 (incorporated by reference herein in its entirety) arecontemplated for use in the compositions and methods disclosed hereinand include, but are not limited to, alkylating agents, antibioticagents, antimetabolic agents, hormonal agents, plant-derived agents, andbiologic agents.

Examples of alkylating agents include, but are not limited to,bischloroethylamines (nitrogen mustards, e.g. chlorambucil,cyclophosphamide, ifosfamide, mechlorethamine, melphalan, uracilmustard), aziridines (e.g. thiotepa), alkyl alkone sulfonates (e.g.busulfan), nitrosoureas (e.g. carmustine, lomustine, streptozocin),nonclassic alkylating agents (altretamine, dacarbazine, andprocarbazine), platinum compounds (e.g., carboplastin, cisplatin andplatinum (IV) (Pt(IV))).

Examples of antibiotic agents include, but are not limited to,anthracyclines (e.g. doxorubicin, daunorubicin, epirubicin, idarubicinand anthracenedione), mitomycin C, bleomycin, dactinomycin,plicatomycin. Additional antibiotic agents are discussed in detailbelow.

Examples of antimetabolic agents include, but are not limited to,fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate, leucovorin,hydroxyurea, thioguanine (6-TG), mercaptopurine (6-MP), cytarabine,pentostatin, fludarabine phosphate, cladribine (2-CDA), asparaginase,imatinib mesylate (or GLEEVEC®), and gemcitabine.

Examples of hormonal agents include, but are not limited to, syntheticestrogens (e.g. diethylstibestrol), antiestrogens (e.g. tamoxifen,toremifene, fluoxymesterol and raloxifene), antiandrogens (bicalutamide,nilutamide, flutamide), aromatase inhibitors (e.g., aminoglutethimide,anastrozole and tetrazole), ketoconazole, goserelin acetate, leuprolide,megestrol acetate and mifepristone.

Examples of plant-derived agents include, but are not limited to, vincaalkaloids (e.g., vincristine, vinblastine, vindesine, vinzolidine andvinorelbine), podophyllotoxins (e.g., etoposide (VP-16) and teniposide(VM-26)), camptothecin compounds (e.g., 20(S) camptothecin, topotecan,rubitecan, and irinotecan), taxanes (e.g., paclitaxel and docetaxel).

Chemotherapeutic agents contemplated for use include, withoutlimitation, alkylating agents including: nitrogen mustards, such asmechlor-ethamine, cyclophosphamide, ifosfamide, melphalan andchlorambucil; nitrosoureas, such as carmustine (BCNU), lomustine (CCNU),and semustine (methyl-CCNU); ethylenimines/methylmelamine such asthriethylenemelamine (TEM), triethylene, thiophosphoramide (thiotepa),hexamethylmelamine (HMM, altretamine); alkyl sulfonates such asbusulfan; triazines such as dacarbazine (DTIC); antimetabolitesincluding folic acid analogs such as methotrexate and trimetrexate,pyrimidine analogs such as 5-fluorouracil, fluorodeoxyuridine,gemcitabine, cytosine arabinoside (AraC, cytarabine), 5-azacytidine,2,2′-difluorodeoxycytidine, purine analogs such as 6-mercaptopurine,6-thioguanine, azathioprine, 2′-deoxycoformycin (pentostatin),erythrohydroxynonyladenine (EHNA), fludarabine phosphate, and2-chlorodeoxyadenosine (cladribine, 2-CdA); natural products includingantimitotic drugs such as paclitaxel, vinca alkaloids includingvinblastine (VLB), vincristine, and vinorelbine, taxotere, estramustine,and estramustine phosphate; epipodophylotoxins such as etoposide andteniposide; antibiotics such as actimomycin D, daunomycin (rubidomycin),doxorubicin, mitoxantrone, idarubicin, bleomycins, plicamycin(mithramycin), mitomycinC, and actinomycin; enzymes such asL-asparaginase; biological response modifiers such as interferon-alpha,IL-2, G-CSF and GM-CSF; miscellaneous agents including platinumcoordination complexes such as cisplatin, Pt(IV) and carboplatin,anthracenediones such as mitoxantrone, substituted urea such ashydroxyurea, methylhydrazine derivatives including N-methylhydrazine(MIH) and procarbazine, adrenocortical suppressants such as mitotane(o,p′-DDD) and aminoglutethimide; hormones and antagonists includingadrenocorticosteroid antagonists such as prednisone and equivalents,dexamethasone and aminoglutethimide; progestin such ashydroxyprogesterone caproate, medroxyprogesterone acetate and megestrolacetate; estrogen such as diethylstilbestrol and ethinyl estradiolequivalents; antiestrogen such as tamoxifen; androgens includingtestosterone propionate and fluoxymesterone/equivalents; antiandrogenssuch as flutamide, gonadotropin-releasing hormone analogs andleuprolide; and non-steroidal antiandrogens such as flutamide.

Additional therapeutic agents contemplated by the present disclosureinclude, without limitation, the therapeutic agents in Table 2, below.

TABLE 2 Abacavir Sulfate Abbo-Code Index Abciximab AbobotulinumtoxinaAcamprosate Calcium Accolate Tablets Accutane CapsulesF AcebutololHydrochloride Acetadote Injection Acetaminophen AcetylcysteineAcetylsalicyclic Acid Achillea Millefolium Aciphex Tablets AcitretinAconitum Napellus Acticin Cream Actidose With Sorbitol Actidose-AquaActimmune Suspension Suspension Activase I.V. Active Calcium TabletsActivella Tablets Actonel Tablets Actoplus Met Tablets Actos TabletsAcyclovir Aczone Gel 5% Adalimumab Adcirca Tablets Adefovir DipivoxilAdenocard IV Injection Adenoscan Adenosine Adipex-P Capsules Adipex-PTablets Advair Diskus 100/50 Advair Diskus 250/50 Advair Diskus 500/50Advate Advicor Tablets Afinitor Tablets Aggrenox Capsules Ala(Alpha-Linolenic Acid) Albendazole Albenza Tablets Albumin (Human)Albutein 5% Solution Albutein 25% Solution Albuterol Albuterol SulfateAldara Cream, 5% Aldesleukin Alefacept Alendronate Sodium Alferon NInjection Alfuzosin Hydrochloride Alimta For Injection AliskirenAlitretinoin Alkeran For Injection Alkeran Tablets Allantoin AllegraTablets Allegra-D 12 Hour Allegra-D 24 Hour Allium Cepa AllopurinolExtended-Release Tablets Extended-Release Tablets Almotriptan MalateAloxi Injection Alpha Tocopherol Alpha-Hydroxy Acetate Alpha₁-ProteinaseAlphagan P Ophthalmic Alphanate Alphanine SD Inhibitor (Human) SolutionAlprazolam Altabax Ointment Alteplase Altretamine Aluminum HydroxideAlvimopan Amantadine Ambien Tablets Hydrochloride Ambien CR TabletsAmbisome for Injection Ambrisentan Amerge Tablets Amevive Amicar 500 MGTablets Amicar 1000 MG Tablets Amiloride Hydrochloride Amino AcidPreparations Aminobenzoate Aminohippurate Sodium Aminosalicyclic AcidPotassium 4-Amino-Salicyclic Acid 5-Amino-Salicyclic Acid AmitizaCapsules Amitriptyline Hydrochloride Amlactin Moisturizing Amlactin XLAmlodipine Besylate Amnesteem Capsules Lotion and Cream MoisturizingLotion Amoxicillin Amoxil Capsules Amoxil Tablets Amphotericin B,Liposomal Amrix Capsules Anagrelide Anakinra Ananas ComosusHydrochloride Anaprox Tablets Anaprox DS Tablets Androgel AngeliqTablets Angiomax for Injection Animi-3 Capsules Anthihemophilic FactorAntihemophilic Factor (Human) (Recombinant) Anti-Inhibitor CoagulantAntithrombin Antivenin (Black Widow Anzemet Injection Complex SpiderAntivenin) Anzemet Tablets Apidra Injection Apidra Solostar AplenzinExtended- Injection Release Tablets Appearex Tablets Aprepitant AprisoCapsules Aralast NP Solvent Aranesp for Injection Arcalyst forArgatroban Aricept Tablets Subcataneous Injection Aricept ODT TabletsArixtra Injection Armodafinil Arnica Montana Aromasin Tablets ArranonInjection Arsenic Trioxide Artemether Asacol Delayed-Release Asacol HDDelayed- Ascorbic Acid Asenapine Tablets Release Tablets AsmanexTwisthaler Asparaginase Aspirin Atacand Tablets Atacand HCT 16-12.5Atacand HCT 32-12.5 Atenolol Atomoxetine Tablets Tablets HydrochlorideAtopiclair Cream Atorvastatin Calcium Atovaquone Atripla Tablets AtriplaTablets Atropine Sulfate Atryn Lyophilized Attenuvax Powder AugmentinTablets Augmentin XR Extended Authia Cream Avalide Film-Coated ReleaseTablets Tablets Avalide Tablets Avandamet Tablets Avandaryl TabletsAvandia Tablets Avapro Tablets Avastin IV Avelox I.V. Avelox TabletsAvinza Capsules Avita Cream Avita Gel Avobenzone Avocado Oil AvodartSoft Gelatin Axert Tablets Axid Capsules Capsules Azasite OphthalmicAzelaic Acid Azilect Tablets Azithromycin Drops Azmacort Inhalation AzorTablets Baclofen Balsalazide Disodium Aerosol Balsam Peru Banzel TabletsBasiliximab Bayer Aspirin Bayer Children's Low BOG, Live (intravesical)Beclomethasone Beclomethasone Dose Aspirin Regimen DipropionateDipropionate (81 MG) Chewable Monohydrate Cherry and Orange Beconase AQNasal Bee Pollen Beelith Tablets Belladonna Spray Belladonna AlkaloidsBellis Perennis Benadryl Allergy Ultratab Benazepril HydrochlorideTablets Bendamustine Bendroflumethiazide Benefix Vials Benicar TabletsHydrochloride Benicar HCT Tablets Bentoquatam Bentyl Capsules BentylInjection Bentyl Syrup Bentyl Tablets Benzoyl Peroxide Benzyl AlcoholBesifloxacin Beta-Carotene Betamethasone Betamethasone DipropionateBetamethasone Valerate Betaseron For SC Betimol Ophthalmic BevacizumabInjection Solution Bevitamel Tablets Bexarotene Bexxar Biaxin FilmtabTablets Biaxin Granules Biaxin XL Filmtab Bicalutamide Bicillin C-RInjectable Tablets Bicillin L-A Injection Bilberry Bimatoprost Bio-CTablets Bioflavonoids Biotin Bisacodyl Bismuth Subcitrate PotassiumBisoprolol Fumarate Bivalirudin Black Widow Spider Boniva TabletsAntivenin (Equine) Boostrix Vaccine Boron Bortezomib Bosentan Botox forInjection Botulinum Toxin Type A Brevibloc Injection BrimonidineTartrate Bromelain Bromocriptine Mesylate Budesonide BumetanideBupropion Hydrochloride Buspirone Hydrochloride Busulfan ButenafineHydrochloride Butorphanol Tartrate Byetta Injection Bystolic TabletsCalcijex Injection Calcipotriene Calcitriol Calcium Calcium AscorbateCalcium Carbonate Calcium Citrate Calcium Pantothenate CalendulaPantothenate Caledula Officinalis Camellia Sinensis Campral TabletsCanakinumab Canasa Rectal Cancidas For Injection Candesartan CilexetilCapastat Sulfate for Suppositories Injection Capecitabine CapreomycinSulfate Capryloyl Glycine Captopril Carac Cream 0.5% Carafate SuspensionCarafate Tablets Carbamazepine Carbatrol Capsules Carbidopa CarbolicAcid Cardio Basics Tablets Cardioessentials Capsules Cardizem LaExtended Carica Papaya Carotenoids Release Tablets Carvedilol CarvedilolPhosphate Caspofungin Acetate Castor Oil Catapres-TTS Cathflo ActivaseCefdinir Cefixime Ceftazidime Ceftin Tablets Ceftriaxone SodiumCefuroxime Cefuroxime Axetil Celebrex Capsules Celecoxib Celexa TabletsCephaelis Ipecacuanha Certolizumbab Pegol Cervidil Vaginal InsertCetirizine Hydrochloride Cetrorelix Acetate Cetrotide for InjectionCevimeline Chamomilla Hydrochloride Chantix Tablets Charcoal, ActivatedChelated Mineral Tablets Chemet Capsules Chloral Hydrate ChlorambucilChlordiazepoxide Chlorothiazide Chlorothiazide Sodium ChloroxylenolChlorpheniramine Chlorpheniramine Maleate Polistriex ChlorpropamideChlorthalidone Cholecalciferol Choline Bitartrate ChoriogonadotropinAlfa Chromium Chromium Picolinate Chromium Polynicotinate ChymotrypsinCialis Tablets Cilastatin Sodium Cilostazol Cimetidine Cimetidine CimziaCinacalcet Hydrochloride Hydrochloride Ciprofloxacin CiprofloxacinCisatracurium Besylate Citalopram Hydrochloride Hydrobromide Citranatal90 DHA Citranatal Assure Citranatal Harmony Citrantal RX TabletsCapsules Citric Acid Cladribine Clarinex Tablets Clarinex ReditabsTablets Clarinex-D 12-Hour Clarinex-D 24-Hour Clarithromycin ClavulanatePotassium Extended-Release Tablets Extended-Release Tablets ClevidipineButryate Cleviprex Climara Transdermal Climara Pro Transdermal SystemSystem Clindamycin Clindamycin Phosphate Clinoril Tablets ClobetasolPropionate Clofarabine Clorlar for Intravenous Clomipramine ClonazepamInfusion Hydrochloride Clonidine Clonidine Hydrochloride ClopidogrelBisulfate Clorazepate Dipotassium Clorpactin WCS-90 Clorpres TabletsClotrimazole Clozapine CM Plex Cream CM Plex Softgels Coagulation FactorVIIA, Coartem Tablets Recombinant Cod Liver Oil Codeine PhosphateCoenzyme Q-10 Colesevelam Hydrochloride Collagen Collagenase ColocynthisColostrum Combigan Ophthalmic Combivir Tablets Comtan Tablets ComvaxSolution Concept DHA Prenatal Concept OB Prenatal Concerta Extended-Copaxone for Injection Multivitamin Multivitamin Release TabletsSupplements Supplements Copper Copper, Intrauterine Coquinone 30Capsules Cordymax CS-4 Capsules Coreg Tablets Coreg CR Extended-Correctol Delayed- Cosmegen for Injection Release Capsules ReleaseTablets, USP Cozaar Tablets Creon Delayed-Release Crestor TabletsCrixivan Capsules Capsules Cubicin for Injection Cupric Oxide CuprimineCapsules Cyclobenzaprine Hydrochloride Cycloserine Cyclosporine CymbaltaDelayed- Cysteine Cytomel Tablets Release Capsules Dacogen InjectionDactinomycin D-Alpha Tocopherol Dalteparin Sodium Dapsone DaptomycinDaraprim Tablets Darbepoetin Alfa Darifenacin Darvocet-A 500 TabletsDarvocet-N 50 Tablets Darvocet-N 100 Tablets Darvon Pulvules Darvon-NTablets Daytrana Transdermal Ddrops Dietary Patch Supplement DecitabineDeferasirox Delatestryl Injection Demser Capsules Denavir CreamDenileukin Diftitox Depakene Capsules Depakote Delayed Release TabletsDepakote ER Extended Depakote Sprinkle Deprenyl Derma-Smoothe/FS ReleaseTablets Capsules Topical Oil Dermotic Oil Desflurane DesloratadineDesonide Desvenlafaxine Succinate Dexamethasone Dexedrine SpansuleDexlansoprazole Sustained-Release Capsules DexmethylphenidateDextroamphetamine Dextromethorphan Dextrose Hydrochloride SulfateHydrobromide DHA (Docosahexaenoic Diazepam Diazoxide Dibasic SodiumAcid) Phosphate Dibenzyline Capsules Diclofenac Epolamine DiclofenacPotassium Diclofenac Sodium Dicyclomine Didronel Tablets DietarySupplement Digestive Enzymes Hydrochloride Digibind for InjectionDigoxin Immune Fab Dilaudid Injection Dilaudid Tablets Digoxin (Ovine)Dilaudid-HP Injection Dilaudid-HP Lyophilized Diltiazem HydrochlorideDinoprostone Powder 250 MG Dioctyl Sodium Diovan Tablets Diovan HCTTablets Diphenhydramine Sulfosuccinate Hydrochloride DiphenoxylateDiphenylhydantoin Diphtheria & Tetanus Diphtheria and TetanusHydrochloride Toxoids and Acellular Toxoids and Acellular PertussisVaccine Pertussis Adsorbed and Adsorbed Inactivated Poliovirus VaccineDipyridamole Disocorea Divalproex Sodium Divigel Divista SoftgelCapsules Docetaxel Docosahexanenoic Acid Docusate Sodium (DHA)Dolasetron Mesylate Donepezil Hydrochloride Donnatal Extentabs DoribaxInjection Dornase Alfa Doryx Delayed-Release Dorzolamide DoxazosinMesylate Tablets Hydrochloride Doxepin Hydrochloride Doxil InjectionDoxorubicin Doxycycline Hydrochloride Liposome Doxycycline HyclateDronedarone Drospirenone Drotrecogin Alfa (Activated) Duet Tablets DuetDHA Tablets and Duetact Tablets Duloxetine Softgel CapsulesHydrochloride Duraclon Injection Dutasteride Dyazide Capsules DynacircCR Controlled Release Tablets Dyrenium Capsules Dysport for InjectionEchinacea Angustifolia Echinacea Purpurea EC-Naprosyn Delayed-Eculizumab Edecrin Tablets Edecrin Sodium Release Tablets IntravenousEdetate Calcium E.E.S. 400 Filmtab E.E.S. Granules Efavirenz DisodiumTablets Effexor XR Extended- Effient Tablets Effient TabletsEicosapentaenoic Acid Release Capsules (EPA) Eldepryl Capsules ElidelCream 1% Eligard 7.5 MG Eligard 22.5 MG Eligard 30 MG Eligard 45 MGElitek Elmiron Capsules Eloxatin for Injection Elspar for InjectionElspar for Injection Eltrombopag Embeda Extended Emend Capsules Emendfor Injection Emtricitabine Release Capsules Emtriva Capsules EmtrivaOral Solution Enablex Extended- Enalapril Maleate Release Tablets Enbrelfor Injection Enflurane Engerix-B Vaccine Enjuvia Tablets EnoxaparinSodium Entacapone Entereg Capsules Enzymes, Collagenolytic Enzymes,Debridement Enzymes, Digestive Enzymes, Proteolytic EpinastineHydrochloride Epinephrine Epipen Auto-Injector Epipen 2-Pak Epipen Jr.Auto-Injector Epipen Jr. 2-Pak Epivir Oral Solution Epivir TabletsEpivir-HBV Oral Solution Epivir-HBV Tablets Epoetin Alfa Epogen forInjection Epoprostenol Sodium Eprosartan Mesylate Eptifibatide EpzicomTablets Equetro Extended- Release Capsules Erlotinib Ertapenem Eryped200 & Eryped 400 Erthromycin Oral Suspension Ethylsuccinate EscitalopramOxalate Esmolol Hydrochloride Esomeprazole Magnesium Esomeprazole SodiumEntrace Tablets Estradiol Estradiol Acetate Estrogens, Conjugated,Synthetic B Estropipate Estrostep FE Tablets Etanercept EthacrynateSodium Ethacrynic Acid Ethinyl Estradiol Ethosuximide EditronateDisodium Etoposide Euphrasia Officinalis Everolimus Evista TabletsEvoxac Capsules Exelon Capsules Exemestane Exenatide Exforge TabletsExforge HCT Tablets Exjade Tablets Extavia Kit Ez-Char ActivatedEzetimibe Factor IX (Human) Factor IX Complex Charcoal PelletsFamotidine Fanapt Tablets Faslodex Injection Fatty Acids FebuxostatFeiba VH Felodipine Femara Tablets Femcon FE Tablets Femhrt TabletsFemtrace Tablets Fenofibrate Fenoglide Tablets Fenoprofen CalciumFentanyl Fentanyl Citrate Fentora Tablets Ferralet 90 Tablets Ferralet90 Tablets Ferrous Fumarate Ferrous Fluconate Ferrous SulfateFesoterodine Fumarate Fexofenadine Hydrochloride Fiber Fiber SupplementFilgrastim Finasteride Flebogamma 5% DIF Flecainide Acetate FleetBabylax Fleet Bisacodyl Laxatives Suppositories Fleet Pedia-LaxFlexbumin 25% I.V. Flolan for Injection Flonase Nasal Spray ChewableTablets Florical Capsules Florical Tablets Flovent Diskus 50 MCG FloventDiskus 100 MCG Flovent Diskus 250 MCG Flovent HFA 44 MCG Flovent HFA 110MCG Flovent HFA 250 MCG Inhalation Aerosol Inhalation Aerosol InhalationAerosol Fluarix Vaccine Fludarabine Phosphate Flulaval InjectionFlumazenil Vaccine Flumist Vaccine Fluocinolone Acetonide FluocinonideFluorouracil Fluoxetine Fluoxetine Hydrochloride Fluphenazine FlurazepamHydrochloride Hydrochloride Flurbiprofen Fluticasone Furoate FluticasonePropionate Fluvoxamine Maleate Focalin XR Capsules Folate Folgard OSTablets Folic Acid Follistim AQ Cartridge Follitropin Alfa FollitropinBeta Fondaparinux Sodium Foradil, Aerolizer Forane Liquid for FormadonSolution Formaldehyde Inhalation Formoterol Fumarate Formoterol FumarateFortaz Injection Fortaz for Injection Dihydrate Forteo for InjectionFosamax Tablets Fosamax Plus D Tablets Fosamprenavir CalciumFosaprepitant Foscarnet Sodium Foscavir Injection Fosrenol ChewableDimeglumine Tablets Fragmin Injection Frova Tablets FrovatriptanSuccinate Fulvestrant Furosemide Gabitril Tablets Galantamine GammagardLiquid Gammagard S/D Gamunex Ganoderma Lucinum Gardasil InjectionMushroom Extract Gemcitabine Gemtuzumac Gemzar for Injection GengrafCapsules Hydrochloride Ozogamicin Genotropin Lyophilized Geodon CapsulesGeodon for Injection Glatiramer Acetate Powder Gleevec Tablets GliadelWafer Glimepiride Glipizide Glucagon Glucono-Delta-Lactone GlucosamineSulfate Glutose 15, Glutose 45 (Oral Glucose Gel) Glyburide GlycerinGlyceryl Guaiacolate Glyceryl Trinitrate Glycyrrhestinic Acid GoldensealGolimumab Gonal-F For Injection Gonal-F RFf for Injection Gonal-F RFFPen for Gordochom Solution Granisetron Injection HydrochlorideGuaifenesin Guanfacine Haemophilus B Haldol Injection HydrochlorideConjugate Vaccine Haldol Decanoate Haloperidol Hamamelis VirginianaHappycode Spray Injection Havrix Injection Vaccine Hemin HemocyteTablets Hemofil M Hepatitis A Vaccine, Hepatitis B Vaccine, HEP-ForteCapsules Heplive Softgel Capsules Inactivated Recombinant HepseraTablets Herbals, Multiple Herbals with Minerals Herbals with Vitamins &Minerals Herceptin I.V. Hexalen Capsules Histrelin Acetate HomeopathicFormulation Humalog-Pen and Humatrope Vials and Humira Injection SyringeHumulin 50/50, 100 Kwikpen Cartridges and Pen Units Humulin 70/30 VialHumulin N Vial Humulin R Humulin R (U-500) Hyalgan Solution HycamtinCapsules Hycamtin for Injection Hycet Oral Solution Hydrastis canadensisHydrochlorothiazide Hydrocodone bitartrate Hydrocodone polistirexHydromorphone Hydroxychloroquine Hydroxypropyl cellulose Hyland's calmsforté 4 hydrochloride sulfate kids tablets Hyand's calms forté Hyland'scalms forté Hyland's cold 'n cough 4 Hyland's colic tablets capletstablets kids Hyland's earache drops Hyland's leg cramps PM Hyland's legcramps with Hyland's leg cramps with with quinine tablets quininecaplets quinine tablets Hyland's nerve tonic Hyland's nerve tonicHyland's restful legs Hyland's sniffles 'n caplets tablets tabletssneezes 4 kids tablets Hyland's teething gel Hyland's teething tabletsHyoscine hydrobromide Hyoscyamine sulfate Hypericum perforatum Hyzaar50-12.5 tablets Hyzaar 100-12.5 tablets Hyzaar 100-25 tabletsIbandronate sodium Ibuprofen Ibuprofen Lysine Ilaris InjectionIloperidone Imatinib mesylate Imipenem Imiquimod Imitrex injectionImitrex nasal spray Imitrex tablets Immune globulin intravenous (human)Immunizen capsules Immunocal powder Imodium A-D liquid Imodiummulti-symptom sachets caplets, and EZ chews relief caplets and chewabletablets Implanon implant Indapamide Indinavir sulfate Indocin capsulesIndocin I.V. Indocin oral suspension Indocin suppositories IndomethacinIndomethacin sodium Infanrix injection vaccine Infants' strengthproducts Infliximab trihydrate Influenza virus vaccine Influenza virusvaccine Innopran XL extended Inositol live, intranasal release capsulesInsulin, human (RDNA Insulin aspart, human Insulin aspart, human Insulinaspart protamine, origin) regular human Insulin detemir (RDNA Insulinglargine Insulin glulisine Insulin Lispro, human origin) Insulin lisproprotamine, Insulin, human NPH Insulin, human regular Insulin, humanregular human and human NPH mixture Integra F supplement Integra plussupplement Integra supplement Integrilin injection capsules capsulescapsules Interferon alfa-2B, Interferon alfa-N3 Interferon beta-1AInterferon beta-1B recombinant (human leukocyte derived) Interferongamma-IB Intravenous sodium diuril Intron A for injection Intunivextended release tablets Invanz for injection Invega extended-releaseInvega sustenna Iodine tablets extended-release injectable suspensionIodine I 131 tositumomab Ipratropium bromide Iquix ophthalmic solutionIrbesartan Iron Carbonyl Iron Polysaccharide Isentress TabletsIsocarboxazid complex Isoflurane Isotretinoin Isradipine Ivermectin IvyBlock Janumet Tablets Januvia Tablets Kaletra Oral Solution KaletraTablets Kapidex Delayed Release Kepivance Keppra XR Extended- CapsulesRelease Tablets Ketek Tablets Ketoconazole Ketoprofen KetorolacTromethamine Ketotifen Fumarate Kineret Injection Kinrix InjectionVaccine Klonopin Tablets Klonopin Wafers Klor-Con s/Klor-Con 10 Klor-ConM20/Flor-Con K-Phos Original (Sodium Tablets M10/Klor-Con M15 Free)Tablets Tablets K-Phos M.F. Tablets K-Phos Neutral Tablets K-Phos No. 2Tablets Kristalose for Oral Solution Lacosamide Lacrisert Sterile LacticAcid Lactulose Ophthalmic Insert Lamictal Chewable Lamictal ODT OrallyLamictal Tablets Lamictal XR Extended- Dispersible TabletsDisintegrating Tablets Release Tablets Laminaria Hyperborea LamivudineLamotrigine Lanoxin Injection Lanoxin Injection Lanoxin TabletsLanthanum Carbonate Lantus Injection Pediatric Lapatine L-ArginineL-Carnitine L-Cysteine Lepirudin Letairis Tablets Letrozole LeukeranTablets Leuprolide Acetate Leustatin Injection Levaquin InjectionLevaquin Oral Solution Levaquin Tablets Levaquin in 5% Dextrose LevemirInjection Levetiracetam Injection Levitra Tablets Levitra Tablets (seeLevocarnitine Levocetirizine Schering) Dihydrochloride LevodopaLevofloxacin Levonorgestrel Levothyroxine Sodium Levoxyl Tablets LexaproOral Suspension Lexapro Tablets Lexiscan Injection Lexiva OralSuspension Lexiva Tablets Lialda Tablets Lidocaine Lidoderm PatchLifepak Capsules Linezolid Liothyronine Sodium Lipitor Tablets LipoicAcid Lisdexamfetamine Lisinopril Dimesylate Liver, Dessicated LiverFractions Liver Preparations L-Lysine Loestrin 24 Fe Tablets LoperamideLopinavir Lorazepam Hydrochloride Losartan Potassium LoseasoniqueTablets Lovastatin Lovaza Capsules Lovenox Injection LoxapineHydrochloride L-Proline Lubiprostone Lucentis Injection LumefantrineLumigan Ophthalmic Lupron Depot 3.75 MG Solution Lupron Depot 7.5 MGLupron Depot-3 month Lupron Depot-3 month Lupron Depot-4 month 11.25 MG22.5 MG 30 MG Lupron Depot-Fed 7.5 Lutein Lutropin Alfa Luveris forInjection MG, 11.25 MG and 15 MG Lybrel Tablets Lycium BarbarumLycopodium Clavatum Lyrica Capsules Mafenide Acetate Mag-Al LiquidMag-Al Plus Mag-Al Plus XS Mag-Al Ultimate Magnesium Magnesium CarbonateMagnesium Citrate Strength Magnesium Hydroxide Magnesium Oxide MagnesiumSulfate Malarone Pediatric Tablets Malarone Tablets Manganese ManganeseSulfate Maprotiline Hydrochloride Maraviroc Marineomega Softgel MaritimePine Extract Marplan Tablets Capsules Mavik Tablets Maxair AutohalerMaxalt Tablets Maxalt-MLT Orally Disintegrating Tablets Maximum StrengthMaxzide Tablets Maxzide-25 MG Tablets Measles, Mumps, Products Rubellaand Varicella Virus Vaccine, Live Measles, Mumps & Measles VirusVaccine, Mechlorethamine Meclofenamate Sodium Rubella Virus Vaccine,Live Hydrochloride Live Med Omega Fish Oil Medizym TabletsMedroxyprogesterone Mega Antioxidant Acetate Tablets Megace Es OralMegestrol Acetate Meili Soft Capsules Meili Clear Soft CapsulesSuspension Melatonin Meloxicam Melphalan Melphalan HydrochlorideMemantine Menthol Mephyton Tablets Mepron Suspension HydrochlorideMercaptopurine Meribin Capsules Meridia Capsules Meropenem Merrem I.V.Meruvax II Mesalamine Metadate CD Capsules Metaxalone MetforminHydrochloride Methadone Hydrochloride Methenamine Mandelate MethionneMethotrexate Sodium Methyclothiazide Methyl Salicylate MethyldopaMethylnaltrexone Methylphenidate Methylphenidate Bromide HyrdochlorideMetoclopramide Metolazone Metoprolol Succinate Metoprolol TartrateHydrochloride Metozolov Tablets Metronidazole Metyrosine Mevacor TabletsMicafungin Sodium Micardis Tablets Micardis HCT Tablets MiconazoleNitrate Midodrine Hydrochloride Milk of Magnesia Milk of Magnesia Milkof Magnesia Suspension Concentrate (24% Suspension) Milnacipran MineralOil Minerals Minerals, Multiple Hydrochloride Minocycline MirtazapineMitoxantrone M-M-R II Hydrochloride Hydrochloride Moban Tablets ModafmilModicon Tablets Molindone Hydrochloride Molybdenum Mometasone FuroateMometasone Furoate Monobasic Sodium Monohydrate Phosphate MontelukastSodium Morphine Sulfate Motrin IB Tablets and Children's Motrin DosingCaplets Chart Children's Motrin Oral Children's Motrin Non- Infants'Motrin Infants' Motrin Non- Suspension Staining Dye-Free OralConcentrated Drops Staining Dye-Free Suspension Concentrated DropsJunior Strength Motrin Moviprep Oral Solution Moxatag TabletsMoxifloxacin Caplets and Chewable Hydrochloride Tablets MS ContinTablets Multaq Tablets Multiminerals Multivitamins Multivitamins withMumps Virus Vaccine, Mumpsvax Mupirocin Minerals Live Mupirocin CalciumMuromonab-CD3 Mustargen for Injection Mycamine for InjectionMycophenolate Mofetil Mycophenolic Acid Myfortic Tablets Myleran TabletsMylotarg for Injection Nadolol Naftifine Hydrochloride Nameda OralSolution Nameda Tablets Naprosyn Suspension Naprosyn Tablets NaproxenNaproxen Sodium Naratriptan Nasacort AQ Nasal Spray Nascobal Nasal SprayHydrochloride Nasonex Nasal Spray Natrecor for Injection NaturethroidTablets Nebivolol Nelarabine Nembutal Sodium Neoprofen Injection NeoralOral Solution Solution, USP Neoral Soft Gelatin Neulasta InjectionNeupogen for Injection Nevirapine Capsules Nexium Delayed-Release NexiumDelayed-Release Nexium I.V. Niacin Capsule Oral Suspension NiacinamideNiaspan Extended- Nicardipine Nicotinic Acid Release TabletsHydrochloride Nifedipine Nilotnib Nimbex Injection NisoldipineNitrofurantoin Nitrofurantoin Nitroglycerin Nitrolingual PumpsprayMacrocrystals Monohydrate Nizatidine Norditropin Cartridges Norel SRTablets Norelgestromin Norethindrone Norethindrone Acetate NorflexInjectable Norfloxacin Norgestimate Noroxin Tablets Nortriptyline NorvirOral Solution Hydrochloride Norvir Soft Gelatin Norwegian Cod Liver OilNovantrone for Injection Novolog Injection Capsules Concentrate NovologMix 70/30 Novoseven RT Noxafil Oral Suspension Nplate Nucynta TabletsNu-Iron 150 Capsules Nu-Iron Elixir Nutropin for Injection Nutropin AQInjection Nutropin AQ Nuspin Nutropin AQ Pen Nuvaring InjectionCartridge Nuvigil Tablets Nystatin Octocrylene Octreotide Acetate OfortaTablets Olanzapine Olive Oil Olmesartan Medoxomil Olopatadine OmalizumabOmega-3-Acid Ethyl Omega-3 Acids Hydrochloride Esters Omega-3Polyunsaturates Omegalife-3 Omerprazole Omnicef Capsules SupplementationOmnicef for Oral Onabotulinumtoxina Oncaspar Injection OndansetronSuspension Ondansetron Onglyza Tablets Onion Onsolis Film HydrochlorideOntak Vials Opana Tablets Opana ER Tablets Oramorph SR Tablets OrlistatOrphenadrine Ortho-Cept Tablets Ortho Micronor Tablets HydrochlorideOrtho-Novum Tablets Ortho-Novum 1/50 Ortho Tri-Cyclen LO orthoclone OKT3Sterile Tablets Tablets Solution Ortho-Cyclen Tablets OseltamivirPhosphate Osmoprep Tablets Ovcon 35 Tablets Ovcon 50 Tablets OvidrelPrefilled Syringe Oxaliplatin Oxybenzone For Injection OxybutyninChloride Oxycodone Oxycontin Tablets Oxymetazoline HydrochlorideHydrochloride Oxymorphone Palifermin Paliperidone PalivizumabHydrochloride Palonosetron Pancreatin Pancrelipase Panhematin ForInjection Hydrochloride Panitumumab Pantoprazole Sodium Pantothenate,Calcium Pantothenic Acid Papain Parafon Forte DSC Paricalcitol ParnateTablets Paroxetine Paroxetine Hydrochloride Paser Granules PatadayOphthalmic Solution Patanase Nasal Spray Paxil Oral Suspension PaxilTablets Paxil CR Controlled- Release Tablets Pediarix Vaccine LiquidPedvaxhib PEG-3350 Pegasparagase Pegfilgrastim Peginterferon Alfa-2BPegintron Powder For Pemetrexed Disodium Injection Pemirolast PotassiumPenciclovir PenicillaminePenicillin G Penicillin G Procaine BenzathinePentasa Capsules Pentobarbital Sodium Pentosan PolysulfatePentoxifylline Sodium Pepcid Tablets Maximum Strength Percocet TabletsPercodan Tablets Pepcid AC Tablets Perforomist Inhalation PermethrinPerphenazine Petrolatum, White Solution Phenazopyridine PhenobarbitalPhenol Phenoxybenzamine Hydrochloride Hydrochloride PhenterminePhenylazodiamino Phenylephrine Phenyltoloxamine Citrate HydrochloridePyridine Hydrochloride Hydrochloride Phenytek Capsules Phenytoin SodiumExtended Phenytoin Phosphorus Sodium Capsules Photofrin For InjectionPhytonadione Phytosterols Pilocarpine Hydrochloride PimecrolimusPindolol Pink Bismuth Pioglitazone Hydrochloride Piperacillin SodiumPirbuterol Acetate Piroxicam Pitcher Plant Distillate Plan B One-StepTablets Plasma/Albumin-Free Plavix Tablets Pneumococcal Vaccine,Diphtheria Conjugate Pneumococcal Vaccine, Pneumovax 23 PolicosanolPolifeprosan 20 With Polyvalent Carmustine Poliovirus VaccinePolyethylene Glycol Polysaccharide Iron Porfimer Sodium InactivatedComplex Posaconazole Potaba Capsules Potaba Tablets Potassium PotassiumAcid Potassium Chloride Potassium Citrate Potassium Iodide PhosphatePotassium Phosphate Pramlintide Acetate Prasugrel HydrochloridePravastatin Sodium Prazosin Hydrochloride Prednisolone Sodium PregabalinPremarin Intraveous Phosphate Premarin Tablets Premphase Tablets PremproTablets Prenexa Capsules Prevnar Primaxin I.M. Primaxin I.V. PrinivilTablets Prinzide Tablets Pristiq Extended-Release Proair HFA InhalationProbenecid Tablets Aerosol Prochlorperazine Maleate Procosa II TabletsProcrit For Injection Profilnine SD Proflavanol 90 Tablets ProgesteroneProglycem Capsules Proglycem Suspension Prograf Capsules PrografInjection Proguanil Hydrochloride Prolastin Proleukin For InjectionPromacta Tablets Promethazine Prometrium Capsules Hydrochloride (100 MG,200 MG) Propafenone Propecia Tablets Propoxyphene Propoxyphene NapsylateHydrochloride Hydrochloride Propranolol Propylene Glycol Proquad ProscarTablets Hydrochloride Proteolytic Enzymes Protonix For Delayed- ProtonixDelayed-Release Protonix Release Oral Suspension Tablets ProtopicOintment Proventil HFA Inhalation Provigil Tablets Prozac WeeklyCapsules Aerosol Prozac Pulvules Pseudoephedrine Pseudoephedrine SulfatePulmicort Flexhaler Hydrochloride Pulmozyme Inhalation PulsatillaPratensis Pylera Capsules Pyridium Tablets Solution PyrimethamineQuadrivalent Human Quetiapine Fumarate Quinapril HydrochloridePapillomavirum (Types 6, 11, 16, 18) Recombinant Vaccine Quinine QuixinOphthalmic Qvar Inhalation Aerosol Raberprazole Sodium RaloxifeneRaltegravir Ramelteon Ranexa Extended- Hydrochloride Release TabletsRanibizumab Ranitidine Hydrochloride Ranolazine Rapamune Oral SolutionRapamune Tablets Rasagiline Mesylate Rasburicase Razadyne Oral SolutionRazadyne Tablets Razadyne ER Extended- Rebetol Capsules Rebetol OralSolution Release Capsules Rebif Prefilled Syringe Reclast InjectionRecombinate Recombivax HB For Injection Refacto Vials Refludan ForInjection Regadenoson Regular Strength Products Reishimax GLP CapsulesRelenza Inhalation Relistor Injection Relistor Injection Powder RemeronTablets Remeronsoltab Tablets Remicade For IV Renacidin IrrigationInjection Reopro Vials Requip Tablets Requip XL Tablets RestasisOphthalmic Emulsion Retapamulin Retrovir Capsules Retrovir IV InfusionRetrovir Syrup Retrovir Tablets RH₉ (D) Immune Rhus ToxicodendronRibavirin Globulin (Human) Ribes Nigrum Riboflavin Rifaximin RilonaceptRilutek Tablets Riluzole Risedronate Sodium Risperdal M-Tab RisperdalOral Solution Risperdal Tablets Risperdal Consta Long- RisperidoneActing Injection Ritonavir Rituxan Rituximab Rivastigmine TartrateRizathiptan Benzoate Rocephin Injectable Vials Rocuronium Bromide ExtraStrength Rolaids Softchews Vanilla Crème Romazicon Injection RomiplostimRopinirole Hydrochloride Rosiglitazone Maleate Rosuvastatin CalciumRotarix Oral Suspension Rotateq Rotavirus Vaccine, Live, Oral RotavirusVaccine, Live, Roxanol Oral Solution Roxicodone Oral Solution RoxicodoneTablets Oral, Pentavalent Rozerem Tablets Rubella Virus Vaccine,Rufinamide Rythmol Tablets Live Rythmol SR Extended RyzoltExtended-Release Sabril Oral Solution Sabril Tablets Release CapsulesTablets St. Joseph 81 MG Aspirin Saizen For Injection Salagen TabletsSalmeterol Xinafoate Chewable and Enteric Coated Tablets Salmon OilSalonpas Arthritis Salonpas Pain Relief Sandostatin Injection PatchSandostatin LAR Depot Santyl Collagenase Saphris Tablets SarafemOintment Sarapin Vials Sarraceniaceae Savella Tablets SaxagliptinScopolamine Scopolamine Seasonique Tablets Selegiline HydrobromideSelegiline Hydrochloride Selenium Selzentry Tablets Senna SennosidesSen-Sei-Ro Powder Gold Sensipar Tablets Serevent Diskus SeromycinCapsules Seroquel Tablets Seroquel XR Extended- Serostim For InjectionRelease Tablets Sertraline Hydrochloride Sevoflurane Sheep PlacentaSibutramine Hydrochloride Monohydrate Silicea Silicone Simcor TabletsSimethicone Simponi Injection Simulect For Injection SimvastatinSingulair Tablets Singular Oral Granules Sirolimus Sitagliptin PhosphateSkelaxin Tablets Slo-Niacin Tablets Sodium Sodium Acid Phosphate SodiumAscorbate Sodium Chloride Sodium Citrate Sodium Fluoride SodiumHyaluronate Sodium Oxychlorosene Sodium Phosphate Sodium SulfacetamideSodium Sulfate Solifenacin Succinate Soliris Concentrated SolodynExtended Somatostatin Analogue Solution for Intravenous Release TabletsInfusion Somatropin Somatropin (RDNA Son Formula Tablets SorbitolOrigin) Sore Throat Spray Soriatane Capsules Sotalol Hydrochloride SoyOil Spacer, Inhalation Spiriva Handihaler Spironolactone SpirulinaSpringcode Spray Stalevo Tablets Stavudine Strattera Capsules StriantMucoadhesive Stromectol Tablets Succimer Sucralfate Sudafed 12 HourNasal Sudafed 24 Hour Non- Sudafed Nasal Sudafed PE Nasal DecongestantNon- Drowsy Nasal Decongestant Tablets Decongestant Tablets DrowsyCaplets Decongestant Tablets Children's Sudafed Nasal Children's SudafedPE Sudafed OM Sinus Sulfamethoxazole Decongestant Liquid NasalDecongestant Congestion Moisturizing Liquid Nasal Spray Sulfur SulindacSumatriptan Sumatriptan Succinate Sunitinib Malate Super Omega-3Softgels Supprelin La Implant Suprane Liquid for Inhalation Suprax forOral Suprax Tablets Sutent Capsules Symbicort 80/4.5 SuspensionInhalation Aerosol Symbicort 160/4.5 Symbyax Capsules Symlin InjectionSymlinpen Inhalation Aerosol Symphytum Officinale Synagis IntramuscularSynthroid Tablets Syprine Capsules Solution Systane Ultra LubricantTabloid Tablets Taclonex Ointment Taclonex Scalp Topical Eye DropsSuspension Tacrolimus Tadalafil Tambocor Tablets Tamiflu CapsulesTamiflu Oral Suspension Tamoxifen Citrate Tandem Capsules Tandem DHACapsules Tandem F. Capsules Tandem OB Capsules Tandem Plus CapsulesTapentadol Hydrochloride Tarceva Tablets Targretin Capsules TarkaTablets Tasigna Capsules Taurine Taxotere Injection Tazobactam SodiumTegreen 97 Capsules Concentrate Tekturna Tablets Tekturna HCT TabletsTelithromycin Telmesteine Telmisartan Temazepam Temodar Capsules TemodarInjection Temozolomide Temsirolimus Tenecteplase Tenofovir DisoproxilFumarate Terazol 3 Vaginal Terazosin Hydrochloride TerbinafineTeriparatide Suppositories Hydrochloride Testosterone TestosteroneEnanthate Tetrabenazine Tetracycline Hydrochloride Teveten TabletsTeveten HCT Tablets Tev-Tropin for Injection Theophylline TheophyllineAnhydrous Thiamine Disulfide Thiamine Mononitrate ThioguanineThioridazine Thiothixene Thymus Polypeptide Thyroid HydrochlorideTiagabine Hydrochloride Ticarcillin Disodium Tice BCG TigecyclineTimentin Add-Vantage Timentin Injection Timentin IV Infusion TimentinPharmacy Bulk Galaxy Container Package Timolol Hemihydrate TimololMaleate Timoptic In Ocudose Timoptic Sterile Ophthalmic SolutionTiotropium Bromide Tizanidine Tnkase Tobi Nebulizer Solution forInhalation Tobramycin Tocopheryl Acetate Tolazamide Tolbutamide Tolectin200/400/600 Tolmetin Sodium Topamax Sprinkle Topamax Tablets CapsulesTopiramate Topotecan Hydrochloride Toprol-XL Tablets Torisel InjectionTositumomab Toviaz Extended-Release Tracleer Tablets TramadolHydrochloride Tablets Trandolapril Tranylcypromine Sulfate TrastuzumabTraumeel Ear Drops Traumeel Injection Traumeel Oral Drops Traumeel OralLiquid In Traumeel Tablets Solution Vials Travatan Z OphthalmicTravoprost Treanda For Injection Tretinoin Solution Treximet TabletsTriamcinolone Acetonide Triamterene Tribasic Calcium PhosphateTricitrates Oral Solution Tricitrates SF Oral Tricor Tablets TrientineHydrochloride Solution Trifluoperazine Trihexyphenidyl Trilipix DelayedRelease Trimethoprim Hydrochloride Hydrochloride Capsules TrisenoxInjection Trizivir Tablets Trusopt Sterile Truvada Tablets OphthalmicSolution Trypsin Tussionex Pennkinetic Twinject Auto-Injector Tygacilfor Injection Extended-Release Suspension Tykerb Tablets RegularStrength Tylenol Tylox Capsules Uloric Tablets Tables Ultane Liquid ForUltracet Tablets Ultram Tablets Ultram ER Extended- Inhalation ReleaseTablets Ultrase Capsules Ultrase MT Capsules Undecylenic Acid UniphylTablets Urocit-K Tablets Uroqid-Acid No. 2 Uroxatral Tablets Urso 250Tablets Tablets Urso Forte Tablets Ursodiol Vagifem Tablets ValacyclovirHydrochloride Valcyte Tablets Valcyte For Oral Solution ValganciclovirValium Tablets Hydrochloride Valproic Acid Valrubicin Valsartan ValstarSterile Solution For Intravesical Instillation Valtrex Caplets ValturnaTablets Vanadium Vantas Implant Vaprisol Vaqta Vardenafil HydrochlorideVarenicline Tartrate Varicella Virus Vaccine, Varivax Vectribix VelcadeFor Injection Live Venlafaxine Ventolin HFA Inhalation Veramyst NasalSpray Verapamil Hydrochloride Hydrochloride Aerosol Verteporfin VesicareTablets Vicodin Tablets Vicodin ES Tablets Vicodin HP Tablets VicoprofenTablets Vigabatrin Vigamox Ophthalmic Solution Vimpat Injection VimpatTablets Viokase Powder Viokase Tablets Viramune Oral Viramune TabletsViread Tablets Visudyne For Injection Suspension Visutein CapsulesVitamin A Vitamin B1 Vitamin B2 Vitamin B6 Vitamin B12 Vitamin C VitaminD Vitamin D3 Vitamin E Vitamin K Vitamin K1 Vitamins, Multiple Vitamins,Prenatal Vitamins with Minerals Vitis Vinifera Von Willebrand FactorVorinostat Vytorin 10/10 Tablets Vytorin 10/10 Tablets (Human) Vytorin10/20 Tablets Vytorin 10/40 Tablets Vytorin 10/80 Tablets VyvanseCapsules Watchhaler Welchol Tablets Wellburtrin Tablets Wellbutrin SRSustained- Release Tablets Westhroid Tablets White Petrolatum Winrho SDFXeloda Tablets Xenazine Tablets Xenical Capsules Xifaxan Tablets XigrisPowder For Intravenous Infusion Xolair Xolair Xyntha Vials Xyzal OralSolution Xyzal Oral Solution Xyzal Tablets Xyzal Tablets Yarrow YazTablets Yeast Zafirlukast Zaleplon Zanamivir Zantac 25 Efferdose Zantac150 Tablets Zantac 300 Tablets Tablets Zantac Injection Zantac InjectionZantac Injection Zantac Syrup Pharmacy Bulk Package Premixed ZeaxanthinZeel Injection Solution Zemplar Capsules Zemplar Injection ZemuronInjection Zetia Tablets Zetia Tablets Ziagen Oral Solution ZiagenTablets Zidovudine Zinacef For Injection Zinacef Injection Zinc ZincCitrate Zinc Oxide Zinc Sulfate Zinc-220 Capsules ZiprasidoneZiprasidone Mesylate Zipsor 25 MG Liquid Hydrochloride Filled CapsulesZocor Tablets Zofran Injection Zofran Injection Zofran Oral SolutionPremixed Zofran Tablets Zofran ODT Orally Zoledronic Acid ZolinzaCapsules Disintegrating Tablets Zolmitriptan Zolpidem Tartrate ZometaFor Intravenous Zomig Tablets Infusion Zomig Nasal Spray Zomig-ZMTTablets Zonegran Capsules Zonisamide Zorbtive For Injection ZostavaxInjection Zoster Vaccine Live Zosyn for Injection Zovirax CapsulesZovirax Suspension Zovirax Tablets Zyban Sustained-Release TabletsZydone Tablets Zyprexa Tablets Zyprexa Intramuscular Zyprexa ZydisOrally Disintegrating Tablets Zyrtec Allergy Tablets Zyvox For OralZyvox Injection Zyvox Tablets Suspension

Antibiotic Compositions

In some aspects, the additional agent can be an antibiotic composition,or in other aspects the nanoconjugate itself functions as an antibioticcomposition. Accordingly, in some embodiments the present disclosureprovides antibiotic compositions comprising a nanoconjugate as describedherein. Antibiotic compositions as part of functionalized nanoparticlesare also described in PCT/US2010/020558, which is incorporated herein byreference in its entirety.

In aspects wherein the nanoconjugate comprises a polynucleotide aseither a structural biomolecule or a non-structural additional agent, itis contemplated in certain aspects that the polynucleotide issufficiently complementary to a target coding or non-coding sequence ofa prokaryotic gene that it will hybridize to the target sequence underconditions that allow hybridization. In various embodiments, it iscontemplated that hybridization of the nanoconjugate comprising apolynucleotide to a prokaryotic gene inhibits (or prevents) the growthof a prokaryotic cell. Thus, the hybridization of the nanoconjugatecomprising a polynucleotide to a prokaryotic gene is contemplated toresult in a bacteriostatic or bactericidal effect in aspects wherein theprokaryote is bacteria. In aspects wherein the hybridization occurs invivo, the growth of the prokaryotic cell is inhibited compared to thegrowth of the prokaryotic cell in the absence of contact with thepolynucleotide-modified nanoparticle.

In some embodiments, hybridization of the nanoconjugate comprising apolynucleotide to a prokaryotic gene inhibits expression of a functionalprokaryotic protein encoded by the prokaryotic gene. A “functionalprokaryotic protein” as used herein refers to a full length wild typeprotein encoded by a prokaryotic gene, and in certain aspects, thefunctional protein is essential for prokaryotic cell growth.

Prokaryotic proteins essential for growth include, but are not limitedto, a gram-negative gene product, a gram-positive gene product, cellcycle gene product, a gene product involved in DNA replication, a celldivision gene product, a gene product involved in protein synthesis, abacterial gyrase, and an acyl carrier gene product. These classes arediscussed in detail herein below.

The present disclosure also contemplates an antibiotic compositionwherein hybridization to a target non-coding sequence of a prokaryoticgene results in expression of a protein encoded by the prokaryotic genewith altered activity. In some embodiments, the antibiotic compositionhybridizes to a target non-coding sequence of a prokaryotic gene thatconfers a resistance to an antibiotic. These genes are known to those ofordinary skill in the art and are discussed, e.g., in Liu et al.,Nucleic Acids Research 37: D443-D447, 2009 (incorporated herein byreference in its entirety). In some aspects, hybridization of theantibiotic composition to a target non-coding sequence of a prokaryoticgene that confers a resistance to an antibiotic results in increasingthe susceptibility of the prokaryote to an antibiotic. In one aspect,the susceptibility of the prokaryote to the antibiotic is increasedcompared to the susceptibility of the prokaryote that was not contactedwith the antibiotic composition. Relative susceptibility to anantibiotic can be determined by those of ordinary skill in the art usingroutine techniques as described herein.

Combination Therapy with Antibiotics

In some embodiments, the antibiotic composition comprising thenanoconjugate is formulated for administration in combination with anantibiotic agent, wherein both the nanoconjugate and antibiotic agentare administered in a therapeutically effective amount.

The term “antibiotic agent” as used herein means any of a group ofchemical substances having the capacity to inhibit the growth of, or tokill bacteria, and other microorganisms, used chiefly in the treatmentof infectious diseases. See, e.g., U.S. Pat. No. 7,638,557 (incorporatedby reference herein in its entirety). Examples of antibiotic agentsinclude, but are not limited to, Penicillin G; Methicillin; Nafcillin;Oxacillin; Cloxacillin; Dicloxacillin; Ampicillin; Amoxicillin;Ticarcillin; Carbenicillin; Mezlocillin; Azlocillin; Piperacillin;Imipenem; Aztreonam; Cephalothin; Cefaclor; Cefoxitin; Cefuroxime;Cefonicid; Cefmetazole; Cefotetan; Cefprozil; Loracarbef; Cefetamet;Cefoperazone; Cefotaxime; Ceftizoxime; Ceftriaxone; Ceftazidime;Cefepime; Cefixime; Cefpodoxime; Cefsulodin; Fleroxacin; Nalidixic acid;Norfloxacin; Ciprofloxacin; Ofloxacin; Enoxacin; Lomefloxacin;Cinoxacin; Doxycycline; Minocycline; Tetracycline; Amikacin; Gentamicin;Kanamycin; Netilmicin; Tobramycin; Streptomycin; Azithromycin;Clarithromycin; Erythromycin; Erythromycin estolate; Erythromycin ethylsuccinate; Erythromycin glucoheptonate; Erythromycin lactobionate;Erythromycin stearate; Vancomycin; Teicoplanin; Chloramphenicol;Clindamycin; Trimethoprim; Sulfamethoxazole; Nitrofurantoin; Rifampin;Mupirocin; Metronidazole; Cephalexin; Roxithromycin; Co-amoxiclavuanate;combinations of Piperacillin and Tazobactam; and their various salts,acids, bases, and other derivatives. Anti-bacterial antibiotic agentsinclude, but are not limited to, penicillins, cephalosporins,carbacephems, cephamycins, carbapenems, monobactams, aminoglycosides,glycopeptides, quinolones, tetracyclines, macrolides, andfluoroquinolones.

Biomolecule Markers/Labels

A biomolecule as described herein, in various aspects, optionallycomprises a detectable label. Accordingly, the disclosure providescompositions and methods wherein biomolecule complex formation isdetected by a detectable change. In one aspect, complex formation givesrise to a color change which is observed with the naked eye orspectroscopically.

Methods for visualizing the detectable change resulting from biomoleculecomplex formation also include any fluorescent detection method,including without limitation fluorescence microscopy, a microtiter platereader or fluorescence-activated cell sorting (FACS).

It will be understood that a label contemplated by the disclosureincludes any of the fluorophores described herein as well as otherdetectable labels known in the art. For example, labels also include,but are not limited to, redox active probes, chemiluminescent molecules,radioactive labels, dyes, fluorescent molecules, phosphorescentmolecules, imaging and/or contrast agents as described below, quantumdots, as well as any marker which can be detected using spectroscopicmeans, i.e., those markers detectable using microscopy and cytometry. Inaspects of the disclosure wherein a detectable label is to be detected,the disclosure provides that any luminescent, fluorescent, orphosphorescent molecule or particle can be efficiently quenched by noblemetal surfaces. Accordingly, each type of molecule is contemplated foruse in the compositions and methods disclosed.

Methods of labeling biomolecules with fluorescent molecules andmeasuring fluorescence are well known in the art.

Suitable fluorescent molecules are also well known in the art andinclude without limitation 1,8-ANS (1-Anilinonaphthalene-8-sulfonicacid), 1-Anilinonaphthalene-8-sulfonic acid (1,8-ANS),5-(and-6)-Carboxy-2′,7′-dichlorofluorescein pH 9.0, 5-FAM pH 9.0, 5-ROX(5-Carboxy-X-rhodamine, triethylammonium salt), 5-ROX pH 7.0, 5-TAMRA,5-TAMRA pH 7.0, 5-TAMRA-MeOH, 6 JOE,6,8-Difluoro-7-hydroxy-4-methylcoumarin pH 9.0, 6-Carboxyrhodamine 6G pH7.0, 6-Carboxyrhodamine 6G, hydrochloride, 6-HEX, SE pH 9.0, 6-TET, SEpH 9.0, 7-Amino-4-methylcoumarin pH 7.0, 7-Hydroxy-4-methylcoumarin,7-Hydroxy-4-methylcoumarin pH 9.0, Alexa 350, Alexa 405, Alexa 430,Alexa 488, Alexa 532, Alexa 546, Alexa 555, Alexa 568, Alexa 594, Alexa647, Alexa 660, Alexa 680, Alexa 700, Alexa Fluor 430 antibody conjugatepH 7.2, Alexa Fluor 488 antibody conjugate pH 8.0, Alexa Fluor 488hydrazide-water, Alexa Fluor 532 antibody conjugate pH 7.2, Alexa Fluor555 antibody conjugate pH 7.2, Alexa Fluor 568 antibody conjugate pH7.2, Alexa Fluor 610 R-phycoerythrin streptavidin pH 7.2, Alexa Fluor647 antibody conjugate pH 7.2, Alexa Fluor 647 R-phycoerythrinstreptavidin pH 7.2, Alexa Fluor 660 antibody conjugate pH 7.2, AlexaFluor 680 antibody conjugate pH 7.2, Alexa Fluor 700 antibody conjugatepH 7.2, Allophycocyanin pH 7.5, AMCA conjugate, Amino Coumarin, APC(allophycocyanin), Atto 647, BCECF pH 5.5, BCECF pH 9.0, BFP (BlueFluorescent Protein), BO-PRO-1-DNA, BO-PRO-3-DNA, BOBO-1-DNA,BOBO-3-DNA, BODIPY 650/665-X, MeOH, BODIPY FL conjugate, BODIPY FL,MeOH, Bodipy R6G SE, BODIPY R6G, MeOH, BODIPY TMR-X antibody conjugatepH 7.2, Bodipy TMR-X conjugate, BODIPY TMR-X, MeOH, BODIPY TMR-X, SE,BODIPY TR-X phallacidin pH 7.0, BODIPY TR-X, MeOH, BODIPY TR-X, SE,BOPRO-1, BOPRO-3, Calcein, Calcein pH 9.0, Calcium Crimson, CalciumCrimson Ca2+, Calcium Green, Calcium Green-1 Ca2+, Calcium Orange,Calcium Orange Ca2+, Carboxynaphthofluorescein pH 10.0, Cascade Blue,Cascade Blue BSA pH 7.0, Cascade Yellow, Cascade Yellow antibodyconjugate pH 8.0, CFDA, CFP (Cyan Fluorescent Protein), CI-NERF pH 2.5,CI-NERF pH 6.0, Citrine, Coumarin, Cy 2, Cy 3, Cy 3.5, Cy 5, Cy 5.5,CyQUANT GR-DNA, Dansyl Cadaverine, Dansyl Cadaverine, MeOH, DAPI,DAPI-DNA, Dapoxyl (2-aminoethyl) sulfonamide, DDAO pH 9.0, Di-8 ANEPPS,Di-8-ANEPPS-lipid, DiI, DiO, DM-NERF pH 4.0, DM-NERF pH 7.0, DsRed,DTAF, dTomato, eCFP (Enhanced Cyan Fluorescent Protein), eGFP (EnhancedGreen Fluorescent Protein), Eosin, Eosin antibody conjugate pH 8.0,Erythrosin-5-isothiocyanate pH 9.0, Ethidium Bromide, Ethidiumhomodimer, Ethidium homodimer-1-DNA, eYFP (Enhanced Yellow FluorescentProtein), FDA, FITC, FITC antibody conjugate pH 8.0, FlAsH, Fluo-3,Fluo-3 Ca2+, Fluo-4, Fluor-Ruby, Fluorescein, Fluorescein 0.1 M NaOH,Fluorescein antibody conjugate pH 8.0, Fluorescein dextran pH 8.0,Fluorescein pH 9.0, Fluoro-Emerald, FM 1-43, FM 1-43 lipid, FM 4-64, FM4-64, 2% CHAPS, Fura Red Ca2+, Fura Red, high Ca, Fura Red, low Ca,Fura-2 Ca2+, Fura-2, high Ca, Fura-2, no Ca, GFP (S65T), HcRed, Hoechst33258, Hoechst 33258-DNA, Hoechst 33342, Indo-1 Ca2+, Indo-1, Ca free,Indo-1, Ca saturated, JC-1, JC-1 pH 8.2, Lissamine rhodamine,LOLO-1-DNA, Lucifer Yellow, CH, LysoSensor Blue, LysoSensor Blue pH 5.0,LysoSensor Green, LysoSensor Green pH 5.0, LysoSensor Yellow pH 3.0,LysoSensor Yellow pH 9.0, LysoTracker Blue, LysoTracker Green,LysoTracker Red, Magnesium Green, Magnesium Green Mg2+, MagnesiumOrange, Marina Blue, mBanana, mCherry, mHoneydew, MitoTracker Green,MitoTracker Green FM, MeOH, MitoTracker Orange, MitoTracker Orange,MeOH, MitoTracker Red, MitoTracker Red, MeOH, mOrange, mPlum, mRFP,mStrawberry, mTangerine, NBD-X, NBD-X, MeOH, NeuroTrace 500/525, greenfluorescent Nissl stain-RNA, Nile Blue, EtOH, Nile Red, Nile Red-lipid,Nissl, Oregon Green 488, Oregon Green 488 antibody conjugate pH 8.0,Oregon Green 514, Oregon Green 514 antibody conjugate pH 8.0, PacificBlue, Pacific Blue antibody conjugate pH 8.0, Phycoerythrin, PicoGreendsDNA quantitation reagent, PO-PRO-1, PO-PRO-1-DNA, PO-PRO-3,PO-PRO-3-DNA, POPO-1, POPO-1-DNA, POPO-3, Propidium Iodide, PropidiumIodide-DNA, R-Phycoerythrin pH 7.5, ReAsH, Resorufin, Resorufin pH 9.0,Rhod-2, Rhod-2 Ca2+, Rhodamine, Rhodamine 110, Rhodamine 110 pH 7.0,Rhodamine 123, MeOH, Rhodamine Green, Rhodamine phalloidin pH 7.0,Rhodamine Red-X antibody conjugate pH 8.0, Rhodaminen Green pH 7.0,Rhodol Green antibody conjugate pH 8.0, Sapphire, SBFI-Na+, Sodium GreenNa+, Sulforhodamine 101, EtOH, SYBR Green I, SYPRO Ruby, SYTO 13-DNA,SYTO 45-DNA, SYTOX Blue-DNA, Tetramethylrhodamine antibody conjugate pH8.0, Tetramethylrhodamine dextran pH 7.0, Texas Red-X antibody conjugatepH 7.2, TO-PRO-1-DNA, TO-PRO-3-DNA, TOTO-1-DNA, TOTO-3-DNA, TRITC,X-Rhod-1 Ca2+, YO-PRO-1-DNA, YO-PRO-3-DNA, YOYO-1-DNA, and YOYO-3-DNA.

It is also contemplated by the disclosure that, in some aspects,fluorescent polypeptides are used.

Any detectable polypeptide known in the art is useful in the methods ofthe disclosure, and in some aspects is a fluorescent protein. In someaspects, the fluorescent protein is selected from the list of proteinsin Table 3, below.

TABLE 3 List of fluorescent polypeptides Green Proteins EGFP EmeraldCoralHue ® Azami Green CoralHue ® Monomeric Azami Green CopGFP AceGFPZsGreen1 TagGFP TurboGFP mUKG Blue/UV Proteins EBFP TagBFP Azurite EBFP2mKalama1 GFPuv Sapphire T-Sapphire Cyan Proteins ECFP Cerulean AmCyan1CoralHue ® Midoriishi-Cyan TagCFP mTFP1 Yellow Proteins EYFP CitrineVenus PhiYFP TagYFP Turbo YFP ZsYellow1 Orange Proteins CoralHue ®Kusabira-Orange CoralHue ® Monomeric Kusabira-Orange mOrange mKOκ RedProteins TurboFP602 tdimer2(12) mRFP1 DsRed-Express DsRed2 DsRed-MonomerHcRed1 AsRed2 eqFP611 mRaspberry mCherry mStrawberry mTangerine tdTomatoTagRFP JRed Far Red Proteins TurboFP635 mPlum AQ143 TagFP635HcRed-Tandem Large Stokes Shift Proteins CoralHue ® mKeima RedCoralHue ® dKeima Red CoralHue ® dKeima570 Photoconvertible ProteinsCoralHue ® Dronpa CoralHue ® Kaede (green) CoralHue ® Kaede (red)CoralHue ® KikGR1 (green) CoralHue ® KikGR1 (red) KFP-Red PA-GFP PS-CFPPS-CFP mEosFP mEosFP

Contrast Agents

Disclosed herein are, in various aspects, methods and compositionscomprising a nanoconjugate, wherein the biomolecule is a polynucleotide,and wherein the polynucleotide is conjugated to a contrast agent througha conjugation site. In further aspects, a contrast agent is conjugatedto any other biomolecule as described herein. As used herein, a“contrast agent” is a compound or other substance introduced into a cellin order to create a difference in the apparent density of variousorgans and tissues, making it easier to see the delineate adjacent bodytissues and organs. It will be understood that conjugation of a contrastagent to any biomolecule described herein is useful in the compositionsand methods of the disclosure.

Methods provided by the disclosure include those wherein relaxivity ofthe contrast agent in association with a nanoconjugate is increasedrelative to the relaxivity of the contrast agent in the absence of beingassociated with a nanoparticle. In some aspects, the increase is about1-fold to about 20-fold. In further aspects, the increase is about2-fold fold to about 10-fold, and in yet further aspects the increase isabout 3-fold.

In some embodiments, the contrast agent is selected from the groupconsisting of gadolinium, xenon, iron oxide, a manganese chelate(Mn-DPDP) and copper. Thus, in some embodiments the contrast agent is aparamagnetic compound, and in some aspects, the paramagnetic compound isgadolinium.

The present disclosure also contemplates contrast agents that are usefulfor positron emission tomography (PET) scanning. In some aspects, thePET contrast agent is a radionuclide. In certain embodiments thecontrast agent comprises a PET contrast agent comprising a labelselected from the group consisting of ¹¹C, ¹³N, ¹⁸F, ⁶⁴Cu, ⁶⁸Ge,^(99m)Tc and ⁸²Ru. In particular embodiments the contrast agent is a PETcontrast agent selected from the group consisting of [¹¹C]choline,[¹⁸F]fluorodeoxyglucose(FDG), [¹¹C]methionine, [¹¹C]choline,[¹¹C]acetate, [¹⁸F]fluorocholine, ⁶⁴Cu chelates, ^(99m)Tc chelates, and[¹⁸F]polyethyleneglycol stilbenes.

The disclosure also provides methods wherein a PET contrast agent isintroduced into a polynucleotide during the polynucleotide synthesisprocess or is conjugated to a nucleotide following polynucleotidesynthesis. For example and without limitation, nucleotides can besynthesized in which one of the phosphorus atoms is replaced with ³²P or³³P one of the oxygen atoms in the phosphate group is replaced with ³⁵S,or one or more of the hydrogen atoms is replaced with ³H. A functionalgroup containing a radionuclide can also be conjugated to a nucleotidethrough conjugation sites.

The MRI contrast agents can include, but are not limited to positivecontrast agents and/or negative contrast agents. Positive contrastagents cause a reduction in the T₁ relaxation time (increased signalintensity on T₁ weighted images). They (appearing bright on MRI) aretypically small molecular weight compounds containing as their activeelement Gadolinium, Manganese, or Iron. All of these elements haveunpaired electron spins in their outer shells and long relaxivities. Aspecial group of negative contrast agents (appearing dark on MRI)include perfluorocarbons (perfluorochemicals), because their presenceexcludes the hydrogen atoms responsible for the signal in MR imaging.

The composition of the disclosure, in various aspects, is contemplatedto comprise a nanoconjugate that comprises about 50 to about 2.5×10⁶contrast agents. In some embodiments, the nanoconjugate comprises about500 to about 1×10⁶ contrast agents.

Targeting Moiety

The term “targeting moiety” as used herein refers to any molecularstructure which assists a compound or other molecule in binding orotherwise localizing to a particular target, a target area, enteringtarget cell(^(s)), or binding to a target receptor. For example andwithout limitation, targeting moieties may include proteins, includingantibodies and protein fragments capable of binding to a desired targetsite in vivo or in vitro, peptides, small molecules, anticancer agents,polynucleotide-binding agents, carbohydrates, ligands for cell surfacereceptors, aptamers, lipids (including cationic, neutral, and steroidallipids, virosomes, and liposomes), antibodies, lectins, ligands, sugars,steroids, hormones, and nutrients, may serve as targeting moieties.Targeting moieties are useful for delivery of the nanoconjugate tospecific cell types and/or organs, as well as sub-cellular locations.

In some embodiments, the targeting moiety is a protein. The proteinportion of the composition of the present disclosure is, in someaspects, a protein capable of targeting the composition to target cell.The targeting protein of the present disclosure may bind to a receptor,substrate, antigenic determinant, or other binding site on a target cellor other target site.

Antibodies useful as targeting proteins may be polyclonal or monoclonal.A number of monoclonal antibodies (MAbs) that bind to a specific type ofcell have been developed. Antibodies derived through genetic engineeringor protein engineering may be used as well.

The antibody employed as a targeting agent in the present disclosure maybe an intact molecule, a fragment thereof, or a functional equivalentthereof. Examples of antibody fragments useful in the compositions ofthe present disclosure are F(ab′)₂, Fab′ Fab and Fv fragments, which maybe produced by conventional methods or by genetic or proteinengineering.

In some embodiments, the polynucleotide portion of the nanoconjugate mayserve as an additional or auxiliary targeting moiety. The polynucleotideportion may be selected or designed to assist in extracellulartargeting, or to act as an intracellular targeting moiety. That is, thepolynucleotide portion may act as a DNA probe seeking out target cells.This additional targeting capability will serve to improve specificityin delivery of the composition to target cells. The polynucleotide mayadditionally or alternatively be selected or designed to target thecomposition within target cells, while the targeting protein targets theconjugate extracellularly.

It is contemplated that the targeting moiety can, in variousembodiments, be associated with a nanoconjugate. In aspects wherein thenanoconjugate comprises a nanoparticle, it is contemplated that thetargeting moiety is attached to either the nanoparticle, the biomoleculeor both. In further aspects, the targeting moiety is associated with thenanoconjugate composition, and in other aspects the targeting moiety isadministered before, concurrent with, or after the administration of acomposition of the disclosure.

Short Internal Complementary Polynucleotide (sicPN)

In some aspects, the additional agent is a sicPN. A sicPN is apolynucleotide that associates with a polynucleotide that is part of ananoconjugate, and that is displaced and/or released when a targetpolynucleotide hybridizes to the polynucleotide that is part of thenanoconjugate. In one aspect, the sicPN has a lower binding affinity orbinding avidity for the polynucleotide that is part of the nanoconjugatesuch that association of the target molecule with the polynucleotidethat is part of the nanoconjugate causes the sicPN to be displacedand/or released from its association with the polynucleotide that ispart of the nanoconjugate.

“Displace” as used herein means that a sicPN is partially denatured fromits association with a polynucleotide. A displaced sicPN is still inpartial association with the polynucleotide to which it is associated.“Release” as used herein means that the sicPN is sufficiently displaced(i.e., completely denatured) so as to cause its disassociation from thepolynucleotide to which it is associated. In some aspects wherein thesicPN comprises a detectable marker, it is contemplated that the releaseof the sicPN causes the detectable marker to be detected.

Methods for detecting a target molecule using a sicPN are describedherein below.

Transcriptional Regulators

The present disclosure provides compositions comprising a nanoconjugate.In some aspects, the nanoconjugate comprises a polynucleotide, whereinthe polynucleotide further comprises a transcriptional regulator. Inthese aspects, the transcriptional regulator induces transcription of atarget polynucleotide in a target cell.

A transcriptional regulator as used herein is contemplated to beanything that induces a change in transcription of a mRNA. The changecan, in various aspects, either be an increase or a decrease intranscription. In various embodiments, the transcriptional regulator isselected from the group consisting of a polypeptide, a polynucleotide,an artificial transcription factor (ATF) and any molecule known orsuspected to regulate transcription.

Compositions and methods of the disclosure include those wherein thetranscriptional regulator is a polypeptide. Any polypeptide that acts toeither increase or decrease transcription of a mRNA is contemplated foruse herein. A peptide is also contemplated for use as a transcriptionalregulator.

In some embodiments, the polypeptide is a transcription factor. Ingeneral, a transcription factor is modular in structure and contain thefollowing domains.

DNA-binding domain (DBD), which attach to specific sequences of DNA (forexample and without limitation, enhancer or promoter sequences) adjacentto regulated genes. DNA sequences that bind transcription factors areoften referred to as response elements.

Trans-activating domain (TAD), which contain binding sites for otherproteins such as transcription co-regulators. These binding sites arefrequently referred to as activation functions (AFs) [Wärnmark et al.,Mol. Endocrinol. 17(10): 1901-9 (2003)].

An optional signal sensing domain (SSD) (for example and withoutlimitation, a ligand binding domain), which senses external signals and,in response, transmits these signals to the rest of the transcriptioncomplex, resulting in up- or down-regulation of gene expression. Also,the DBD and signal-sensing domains may, in some aspects, reside onseparate proteins that associate within the transcription complex toregulate gene expression.

Regulator Polynucleotides

In some embodiments, the transcription factor is a regulatorpolynucleotide. In certain aspects, the polynucleotide is RNA, and infurther aspects the regulator polynucleotide is a noncoding RNA (ncRNA).

In some embodiments, the noncoding RNA interacts with the generaltranscription machinery, thereby inhibiting transcription [Goodrich etal., Nature Reviews Mol Cell Biol 7: 612-616 (2006)]. In general, RNAsthat have such regulatory functions do not encode a protein and aretherefore referred to as ncRNAs. Eukaryotic ncRNAs are transcribed fromthe genome by one of three nuclear, DNA-dependent RNA polymerases (PolI, II or III). They then elicit their biological responses through oneof three basic mechanisms: catalyzing biological reactions, binding toand modulating the activity of a protein, or base-pairing with a targetnucleic acid.

ncRNAs have been shown to participate actively in many of the diversebiological reactions that encompass gene expression, such as splicing,mRNA turn over, gene silencing and translation [Storz, et al., Annu.Rev. Biochem. 74: 199-217 (2005)]. Notably, several studies haverecently revealed that ncRNAs also actively regulate eukaryotic mRNAtranscription, which is a key point for the control of gene expression.

In another embodiment, a regulatory polynucleotide is one that canassociate with a transcription factor thereby titrating its amount. Insome aspects, using increasing concentrations of the regulatorypolynucleotide will occupy increasing amounts of the transcriptionfactor, resulting in derepression of transcription of a mRNA.

In a further embodiment, a regulatory polynucleotide is an aptamer.

Coating

The coating can be any substance that is a degradable polymer,biomolecule or chemical that is non toxic. Alternatively, the coatingcan be a bioabsorbable coating. As used herein, “coating” refers to thecomponents, in total, that are deposited on a nanoconjugate. The coatingincludes all of the coated layers that are formed on the nanoconjugate.A “coated layer” is formed by depositing a compound, and more typicallya composition that includes one or more compounds suspended, dissolved,or dispersed, in a particular solution. As used herein, the term“biodegradable” or “degradable” is defined as the breaking down or thesusceptibility of a material or component to break down or be brokeninto products, byproducts, components or subcomponents over time such asminutes, hours, days, weeks, months or years. As used herein, the term“bioabsorbable” is defined as the biologic elimination of any of theproducts of degradation by metabolism and/or excretion.

A non-limiting example of a coating that is a biodegradable and/orbioabsorbable material is a bulk erodible polymer (either a homopolymer,copolymer or blend of polymers) such as any one of the polyestersbelonging to the poly(alpha-hydroxy acids) group. This includesaliphatic polyesters such poly(lactic acid); poly(glycolic acid);poly(caprolactone); poly(p-dioxanone) and poly(trimethylene carbonate);and their copolymers and blends. Other polymers useful as abioabsorbable material include without limitation amino acid derivedpolymers, phosphorous containing polymers, and poly(ester amide). Therate of hydrolysis of the biodegradable and/or bioabsorbable materialdepends on the type of monomer used to prepare the bulk erodiblepolymer. For example, the absorption times (time to complete degradationor fully degrade) are estimated as follows: poly(caprolactone) andpoly(trimethylene carbonate) takes more than 3 years; poly(lactic acid)takes about 2 years; poly(dioxanone) takes about 7 months; andpoly(glycolic acid) takes about 3 months. Absorption rates forcopolymers prepared from the monomers such as poly(lacticacid-co-glycolic acid); poly(glycolic acid-co-caprolactone); andpoly(glycolic acid-co-trimethylene carbonate) depend on the molaramounts of the monomers.

The nanoconjugates may also be administered by other controlled-releasemeans or delivery devices that are well known to those of ordinary skillin the art. These include, for example and without limitation,hydropropylmethyl cellulose, other polymer matrices, gels, permeablemembranes, multilayer coatings (see below), liposomes, or a combinationof any of the above to provide the desired release profile in varyingproportions. Other methods of controlled-release delivery of compoundswill be known to the skilled artisan and are within the scope of theinvention.

Methods Methods of Making a Nanoconjugate

The present disclosure provides strategies for crosslinking biomoleculeson a nanoparticle. In one aspect, the strategy involves the use ofalkyne-bearing ligands. These ligands self assemble on to the surface ofgold nanoparticles, and the alkyne moieties of these ligands areactivated by the gold surface for reaction with nucleophiles presentwithin the ligand shell. This crosslinking reaction is suitable forformation of hollow nanoconjugates with any desired surfacefunctionality. Furthermore, any biomolecule or non-biomolecule that canbe attached to a polyalkyne or monoalkyne moiety will be incorporatedinto this ligand shell or form a ligand shell independently

Biomolecule Crosslinking Poly Alkyne Chemistry

Au(I) and Au(III) ions and their complexes display remarkablealkynophilicity, and have been increasingly recognized as potentcatalysts for organic transformations [Hashmi, Chem. Rev. 107: 3180-3211(2007); Li et al., Chem. Rev. 108: 3239 (2008); Fürstner et al., Angew.Chem. Int. Ed. 46: 3410 (2007); Hashmi et al., Angew. Chem. Int. Ed. 45:7896 (2006)]. Recently, it has been demonstrated that Au(0) surfacesalso adsorb terminal acetylene groups and form relatively densely packedand stable monolayers [Zhang et al., J. Am. Chem. Soc. 129: 4876(2006)]. However, the type of interaction that exists between the alkyneand the gold surface is not well understood.

Moreover, it is not clear whether such interaction makes the acetylenegroup more susceptible to chemical reactions, such as nucleophilicadditions typically observed with ionic gold-alkyne complexes. Bearingmultiple side-arm propargyl ether groups, polymer 1 (Scheme 1) readilyadsorbs onto citrate-stabilized 13 nm AuNPs prepared in an aqueoussolution following the Turkevich-Frens method [Frens, Coll. Polym. Sci.250: 736 (1972)]. Excess polymer is removed by iterative centrifugationand subsequent resuspension steps. The resulting polymer-coated AuNPsexhibit a plasmon resonance at 524 nm characteristic of dispersedparticles, and there is no evidence of aggregation even after 8 weeks ofstorage at room temperature. Therefore, even though 1 is a potentialinter-particle crosslinking agent, it does not lead to aggregation ofthe AuNPs, a conclusion that was corroborated by Dynamic LightScattering (DLS) and electron microscopy (see Examples below).

In one embodiment, the disclosure provides a method for synthesizingnanoconjugates from a linear biomolecule bearing pendant propargyl ethergroups (1), utilizing gold nanoparticles (AuNPs) as both the templatefor the formation of the shell and the catalyst for the crosslinkingreaction (Scheme 1). No additional crosslinking reagents or syntheticoperations are required. The reaction yields well-defined, homogeneoushollow nanoconjugates when the nanoparticle is removed after thebiomolecules are crosslinked.

In one aspect, the biomolecule is a polynucleotide as defined herein. Inthese aspects, it is contemplated that the polynucleotide comprises analkyne. In various embodiments, from 1 to 100 alkyne moieties arepresent on a polynucleotide. In further aspects, from about 5 to about50 alkyne moieties, or about 10 to about 20 alkyne moieties are presenton a polynucleotide. In one aspect, 10 alkyne moieties are present onthe polynucleotide. In further aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100or more alkyne moieties are present on a polynucleotide.

In another embodiment, the alkyne moieties on a polynucleotide are onthe 5′ end. In a further embodiment, the alkyne moieties on apolynucleotide are on the 3′ end. It is contemplated that in someaspects the alkyne moieties represent only a portion of the length of apolynucleotide. By way of example, if a polynucleotide is 20 nucleotidesin length, then it is contemplated that the first 10 nucleotides(counting, in various aspects from either the 5′ or 3′ end) comprise analkyne moiety. Thus, 10 nucleotides comprising an alkyne moiety out of atotal of 20 nucleotides results in 50% of the nucleotides in apolynucleotide being associated with an alkyne moiety. In variousaspects it is contemplated that from about 0.01% to about 100% of thenucleotides in a polynucleotide are associated with an alkyne moiety. Infurther aspects, about 1% to about 70%, or about 2% to about 60%, orabout 5% to about 50%, or about 10% to about 50%, or about 10% to about40%, or about 20% to about 50%, or about 20% to about 40% of nucleotidesin a polynucleotide are associated with an alkyne moiety. In stillfurther aspects it is contemplated that about 1%, about 2%, about 3%,about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%,about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%,about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%,about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%,about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%,about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%,about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%,about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about95%, about 96%, about 97%, about 98%, about 99% or about 100% ofnucleotides in a polynucleotide are associated with an alkyne moiety.

In one aspect, this crosslinking reaction can be utilized in theformation of hollow HDL nanoparticles by using an alkyne moiety and aphospholipid-bearing polymer and APO1A proteins (FIG. 16).

Returning to methods of carrying out the crosslinking using a polyalkyne crosslinking approach, the following steps are involved. First, asolution of nanoparticles is prepared (Nanoparticle preparation step) asdescribed herein (Example 1). The solution is brought into contact witha solution comprising biomolecules comprising a poly-reactive group(Contacting step). Depending on the poly reactive group used, anoptional activation step is included (Activation step). The resultingmixture is then incubated to allow the crosslinking to occur (Incubationstep), and is then isolated (Isolation step). An optional dissolution ofthe nanoparticle core (Dissolution step) is then carried out to create ahollow nanoconjugate. A labeling step is also optionally included(Labeling step).

Nucleophiles contemplated for use by the disclosure include thosedescribed herein. In general, nucleophiles contemplated for use can beclassified into carbon nucleophiles, HX nucleophiles (for example andwithout limitation, HF and HCl), oxygen and sulfur nucleophiles, andnitrogen nucleophiles. Any tandem combination of the above is alsocontemplated.

Nanoparticle Preparation Step. A solution of nanoparticles is preparedas described in Example 1. In the case of poly alkyne crosslinking, agold nanoparticle solution is prepared in one aspect.

Contacting step. Biomolecules of interest, which either comprises apoly-reactive group or are modified to contain a poly-reactive group,are contacted with the nanoparticle solution. As used herein, a polyreactive group can be an alkyne, or the poly reactive group can be alight-reactive group, or a group that is activated upon, for example andwithout limitation, sonication or microwaves. Regardless of the polyreactive group that is used, the solution of biomolecules comprising thepoly reactive groups is contacted with the nanoparticle solution tofacilitate the crosslinking.

In some aspects, and regardless of the crosslinking strategy that isused, the amount of biomolecules to add relates to the property of theresulting nanoconjugate. In general, the disclosure providesnanoconjugates that are either more or less dense, depending on theconcentration of biomolecules used to crosslink to the nanoconjugate. Alower concentration of biomolecules will result in a lower density onthe nanoparticle, which will result in a more porous nanoconjugate.Conversely, a higher concentration of biomolecules will result in ahigher density on the nanoparticle, which will result in a more rigidnanoconjugate. As it pertains to these aspects, a “lower density” isfrom about 2 pmol/cm² to about 100 pmol/cm². Also as pertains to theseaspects, a “higher density” is from about 101 pmol/cm² to about 1000pmol/cm².

Activation Step. In aspects of the disclosure wherein a poly reactivegroup present on a biomolecule and/or non-biomolecule requiresactivation, it is contemplated that an activation step is included inthe methods. In this step, the source of activation is applied and canbe, without limitation, a laser (when the poly reactive group is lightreactive), or sound (when the poly reactive group is activated bysonication), or a microwave (when the poly reactive group is activatedby microwaves).

In some embodiments, the surface itself can activate the poly reactivegroup present on a biomolecule and/or non-biomolecule. In theseembodiments, the activation step is not required.

Incubation Step. Once the solution comprising the biomoleculescomprising poly reactive groups is brought into contact with thenanoparticle solution, the mixture is incubated to allow crosslinking tooccur. Incubation can occur at a temperature from about 4° C. to about50° C. The incubation is allowed to take place for a time from about 1minute to about 48 hours or more. It is contemplated that in someaspects the incubation can occur without regard to length of time.

Isolation Step. The crosslinked nanoconjugate can then be isolated. Forisolation, the mixture is centrifuged, the supernatant is removed andthe crosslinked nanoconjugates are resuspended in an appropriate buffer.In various aspects, more than one centrifugation step may be carried outto further purify the crosslinked nanoconjugates.

Dissolution Step. In any of the compositions or methods describedherein, whether to retain the nanoparticle following the crosslinking ofthe biomolecules is optional and dependent of the intended use.

In those embodiments wherein a composition of the disclosure does notcomprise a nanoparticle, it is contemplated that the nanoparticle isdissolved or otherwise removed following the crosslinking of thebiomolecules to the nanoconjugate.

Dissolution of a nanoparticle core is within the ordinary skill in theart, and in one aspect is achieved by using KCN in the presence ofoxygen. In further aspects, iodine or Aqua regia is used to dissolve ananoparticle core. In one aspect, the nanoparticle core comprises gold.As described herein, when KCN is added to citrate stabilized AuNPs, thecolor of the solution changes from red to purple, resulting from thedestabilization and aggregation of the AuNPs. However, for apolymer-coated AuNP, the color slowly changes to a slightly reddishorange color during the dissolution process until the solution is clear(FIG. 9A).

The dissolution process can be visualized by transmission electronmicroscopy (TEM) (FIG. 9C). As the outer layer of the AuNP is partiallydissolved, the protective shell mentioned above can be observed withuranyl-acetate staining of the TEM grid. Complete removal of thetemplate affords hollow nanoconjugates that retain the size and shape oftheir template in high fidelity.

Additional Crosslinking Methods

Direct strand crosslinking (DSC) is a method whereby one or morenucleotides of a polynucleotide is modified with one or morecrosslinking moieties that can be cross-linked through chemical means.The DSC method, in one aspect, is effected through the modification ofone or more nucleotides of a polynucleotide with a moiety that can becrosslinked through a variety of chemical means. In various aspects, theone or more nucleotides that comprise the crosslinking moieties are inthe spacer.

In an aspect, polynucleotides are synthesized that incorporate anamine-modified thymidine phosphoramidite (TN) into the spacer. Thepolynucleotide can consist entirely of this modified base to maximizecross-linking efficiency. The strands are crosslinked in one aspect withthe use of a homobifunctional cross-linker like Sulfo-EGS, which has twoamine reactive NHS-ester moities. Although amines are contemplated foruse in one embodiment, this design is compatible with many otherreactive groups (for example and without limitation, amines, amides,alcohols, esters, aldehydes, ketones, thiols, disulfides, carboxylicacids, phenols, imidazoles, hydrazines, hydrazones, azides, andalkynes).

An additional method, called surface assisted crosslinking (SAC),comprises a mixed monolayer of modified nucleic acids and reactivethiolated molecules that are assembled on the nanoparticle surface andcrosslinked together.

The chemical that causes crosslinking of the biomolecules of interestare known to those of skill in the art, and include without limitationDisuccinimidyl glutarate, Disuccinimidyl suberate,Bis[sulfosuccinimidyl] suberate, Tris-succinimidyl aminotriacetate,succinimidyl 4-hydrazinonicotinate acetone hydrazone, succinimidyl4-hydrazidoterephthalate hydrochloride, succinimidyl 4-formylbenzoate,Dithiobis[succinimidyl propionate],3,3′-Dithiobis[sulfosuccinimidylpropionate], Disuccinimidyl tartarate,Bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone, Ethylene glycolbis[succinimidylsuccinate], Ethylene glycolbis[sulfosuccinimidylsuccinate], Dimethyl adipimidate.2 HCl, Dimethylpimelimidate.2 HCl, Dimethyl Suberimidate.2 HCl,1,5-Difluoro-2,4-dinitrobenzene,β-[Tris(hydroxymethyl)phosphino]propionic acid, Bis-Maleimidoethane,1,4-bismaleimidobutane, Bismaleimidohexane, Tris[2-maleimidoethyl]amine,1,8-Bis-maleimido-diethyleneglycol,1,11-Bis-maleimido-triethyleneglycol, 1,4bismaleimidyl-2,3-dihydroxybutane, Dithio-bismaleimidoethane,1,4-Di-[3′-(2′-pyridyldithio)-propionamido]butane,1,6-Hexane-bis-vinylsulfone,Bis-[b-(4-Azidosalicylamido)ethyl]disulfide, N-(a-Maleimidoacetoxy)succinimide ester, N-[β-Maleimidopropyloxy]succinimide ester,N-[g-Maleimidobutyryloxy]succinimide ester,N-[g-Maleimidobutyryloxy]sulfosuccinimide ester,m-Maleimidobenzoyl-N-hydroxysuccinimide ester,m-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester, Succinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate, Sulfosuccinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate,N-e-Maleimidocaproyloxy]succinimide ester,N-e-Maleimidocaproyloxy]sulfosuccinimide ester, Succinimidyl4-[p-maleimidophenyl]butyrate, Sulfosuccinimidyl4-[p-maleimidophenyl]butyrate,Succinimidyl-6-[β-maleimidopropionamido]hexanoate,Succinimidyl-4-[N-Maleimidomethyl]cyclohexane-1-carboxy-[6-amidocaproate],N-[k-Maleimidoundecanoyloxy]sulfosuccinimide ester, N-Succinimidyl3-(2-pyridyldithio)-propionate, Succinimidyl6-(3-[2-pyridyldithio]-propionamido)hexanoate,4-Succinimidyloxycarbonyl-methyl-a-[2-pyridyldithio]toluene,4-Sulfosuccinimidyl-6-methyl-a-(2-pyridyldithio)toluamido]hexanoate),N-Succinimidyl iodoacetate, Succinimidyl 3-[bromoacetamido]propionate,N-Succinimidyl[4-iodoacetyl]aminobenzoate,N-Sulfosuccinimidyl[4-iodoacetyl]aminobenzoate,N-Hydroxysuccinimidyl-4-azido salicylic acid,N-5-Azido-2-nitrobenzoyloxysuccinimide,N-Hydroxysulfosuccinimidyl-4-azidobenzoate,Sulfosuccinimidyl[4-azidosalicylamido]-hexanoate,N-Succinimidyl-6-(4′-azido-2′-nitrophenylamino) hexanoate,N-Sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino) hexanoate,Sulfosuccinimidyl-(perfluoroazidobenzamido)-ethyl-1,3′-dithioproprionate,Sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3′-proprionate,Sulfosuccinimidyl2-[7-amino-4-methylcoumarin-3-acetamido]ethyl-1,3′dithiopropionate,Succinimidyl 4,4′-azipentanoate, Succinimidyl6-(4,4′-azipentanamido)hexanoate, Succinimidyl2-([4,4′-azipentanamido]ethyl)-1,3′-dithioproprionate, Sulfosuccinimidyl4,4′-azipentanoate, Sulfosuccinimidyl 6-(4,4′-azipentanamido)hexanoate,Sulfosuccinimidyl 2-([4,4′-azipentanamido]ethyl)-1,3′-dithioproprionate,Dicyclohexylcarbodiimide, 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimideHydrochloride, N-[4-(p-Azidosalicylamido)butyl]-3′-(2′-pyridyldithio)propionamide, N-[β-Maleimidopropionic acid]hydrazide, trifluoroacetic acid salt, [N-e-Maleimidocaproicacid]hydrazide, trifluoroacetic acid salt,4-(4-N-Maleimidophenyl)butyric acid hydrazide hydrochloride,N-[k-Maleimidoundecanoic acid]hydrazide, 3-(2-Pyridyldithio)propionylhydrazide, p-Azidobenzoyl hydrazide, N-[p-Maleimidophenyl]isocyanate,and Succinimidyl-[4-(psoralen-8-yloxy)]-butyrate.

DSC and SAC crosslinking of biomolecules has been generally discussedabove. Steps of the methods for these crosslinking strategies willlargely mirror those recited above for poly alkyne crosslinking, exceptthe activation step will not be optional for these crosslinkingstrategies. As described herein, a chemical is used to facilitate thecrosslinking of biomolecules. Thus, a nanoparticle preparation step, acontacting step, activation step, incubation step, isolation step andoptional dissolution step are carried out. A labeling step is optionallyincluded as well. These steps have been described herein above.

The above methods also optionally include a step wherein thenanoconjugates further comprise an additional agent as defined herein.The additional agent can, in various aspects be added to the mixtureduring crosslinking of the biomolecules and/or non-biomolecules, or canbe added after production of the nanoconjugate.

Attachment of a Therapeutic Agent

The disclosure provides, in some embodiments, nanoconjugate compositionswherein the composition further comprises a therapeutic agent. Thetherapeutic agent is, in some aspects, attached to a biomolecule that ispart of the nanoconjugate composition. In further aspects, thebiomolecule is a polynucleotide. Methods of attaching a therapeuticagent or a chemotherapeutic agent to a polynucleotide are known in theart, and are described in Priest, U.S. Pat. No. 5,391,723, Arnold, Jr.,et al., U.S. Pat. No. 5,585,481, Reed et al., U.S. Pat. No. 5,512,667and PCT/US2006/022325, the disclosures of which are incorporated hereinby reference in their entirety.

It will be appreciated that, in various aspects, a therapeutic agent asdescribed herein is attached to the nanoparticle.

Methods Of Using A Nanoconjugate Methods of Using a Hollow Nanoconjugate

Hollow nanoconjugates are useful, in some embodiments, as a deliveryvehicle. Thus, a hollow nanoconjugate is made wherein, in one aspect, anadditional agent as defined herein is localized inside thenanoconjugate. In related aspects, the additional agent is associatedwith the nanoconjugate as described herein. It is contemplated that thenanoconjugate that is utilized as a delivery vehicle is, in someaspects, made more porous, so as to allow placement of the additionalagent inside the nanoconjugate. Porosity of the nanoconjugate can beempirically determined depending on the particular application, and iswithin the skill in the art. All of the advantages of the functionalizednanoparticle (for example and without limitation, increased cellularuptake and resistance to nuclease degradation) are imparted on thehollow nanoconjugate.

It is further contemplated that in some aspects the nanoconjugate usedas a delivery vehicle is produced with a biomolecule that is at leastpartially degradable, such that once the nanoconjugate is targeted to alocation of interest, it dissolves or otherwise degrades in such a wayas to release the additional agent. Biomolecule degradation pathways areknown to those of skill in the art and can include, without limitation,nuclease pathways, protease pathways and ubiquitin pathways.

In some aspects, a composition of the disclosure acts as asustained-release formulation. In these aspects, the nanoconjugate isproduced using poly-lactic-coglycolic acid (PLGA) polymer due to itsbiocompatibility and wide range of biodegradable properties. Thedegradation products of PLGA, lactic and glycolic acids, can be clearedquickly within the human body. Moreover, the degradability of thispolymer can be adjusted from months to years depending on its molecularweight and composition [Lewis, “Controlled release of bioactive agentsfrom lactide/glycolide polymer,” in: M. Chasin and R. Langer (Eds.),Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: NewYork, 1990), pp. 1-41, incorporated by reference herein in itsentirety].

Methods of Increasing Hybridization Rate

In some embodiments, the biomolecule attached to a nanoparticle is apolynucleotide. Accordingly, methods provided include those that enablean increased rate of association of a polynucleotide with a targetpolynucleotide through the use of a sicPN. The increase in rate ofassociation is, in various aspects, from about 2-fold to about 100-foldrelative to a rate of association in the absence of a sicPN. Accordingto the disclosure, the polynucleotide that associates with the targetpolynucleotide is part of a nanoconjugate. Additionally, a sicPN isadded that overlaps with a portion of the target polynucleotide bindingsite on the polynucleotide used to produce the nanoconjugate, but notthe complete sequence.

Thus, there remains a single stranded portion of the polynucleotide thatis part of the nanoconjugate. When the target polynucleotide associateswith the single stranded portion of the polynucleotide that is part ofthe nanoconjugate, it displaces and/or releases the sicPN and results inan enhanced association rate of the polynucleotide that is part of thenanoconjugate with the target polynucleotide.

The association of the polynucleotide with the target polynucleotideadditionally displaces and, in some aspects, releases the sicPN. ThesicPN or the target polynucleotide, in various embodiments, furthercomprises a detectable label. Thus, in one aspect of a method whereindetection of the target polynucleotide is desired, it is thedisplacement and/or release of the sicPN that generates the detectablechange through the action of the detectable label. In another methodwherein detection of the target polynucleotide is desired, it is thetarget polynucleotide that generates the detectable change through itsown detectable label. In methods wherein inhibition of the targetpolynucleotide expression is desired, it is the association of thepolynucleotide that is part of the nanoconjugate with the targetpolynucleotide that generates the inhibition of target polynucleotideexpression through an antisense mechanism.

The compositions of the disclosure comprise a plurality of sicPNs, ableto associate with a plurality of polynucleotides, that may be used onone or more surfaces to specifically associate with a plurality oftarget polynucleotides. Thus, the steps or combination of steps of themethods described below apply to one or a plurality of polynucleotidesthat are part of one or more nanoconjugates, sicPNs and targetpolynucleotides.

In various aspects, the methods include use of a polynucleotide which is100% complementary to the target polynucleotide, i.e., a perfect match,while in other aspects, the polynucleotide is at least (meaning greaterthan or equal to) about 95% complementary to the polynucleotide over thelength of the polynucleotide, at least about 90%, at least about 85%, atleast about 80%, at least about 75%, at least about 70%, at least about65%, at least about 60%, at least about 55%, at least about 50%, atleast about 45%, at least about 40%, at least about 35%, at least about30%, at least about 25%, at least about 20% complementary to thepolynucleotide over the length of the polynucleotide to the extent thatthe polynucleotide is able to achieve the desired of inhibition of atarget gene product. It will be understood by those of skill in the artthat the degree of hybridization is less significant than a resultingdetection of the target polynucleotide, or a degree of inhibition ofgene product expression.

Methods of Detecting a Target Polynucleotide

The disclosure provides methods of detecting a target biomoleculecomprising contacting the target biomolecule with a composition asdescribed herein. The contacting results, in various aspects, inregulation of gene expression as provided by the disclosure. In anotheraspect, the contacting results in a detectable change, wherein thedetectable change indicates the detection of the target biomolecule.Detection of the detectable label is performed by any of the methodsdescribed herein, and the detectable label can be on a biomolecule thatis part of a nanoconjugate, or can be on the target biomolecule.

In some aspects, and as described above, it is the displacement and/orrelease of the sicPN that generates the detectable change. Thedetectable change is assessed through the use of a detectable label, andin one aspect, the sicPN is labeled with the detectable label. Furtheraccording the methods, the detectable label is quenched when inproximity with a surface used to template the nanoconjugate. While it isunderstood in the art that the term “quench” or “quenching” is oftenassociated with fluorescent markers, it is contemplated herein that thesignal of any marker that is quenched when it is relativelyundetectable. Thus, it is to be understood that methods exemplifiedthroughout this description that employ fluorescent markers are providedonly as single embodiments of the methods contemplated, and that anymarker which can be quenched can be substituted for the exemplaryfluorescent marker.

The sicPN is thus associated with the nanoconjugate in such a way thatthe detectable label is in proximity to the surface to quench itsdetection. When the polynucleotide that is part of the nanoconjugatecomes in contact and associates with the target polynucleotide, itcauses displacement and/or release of the sicPN. The release of thesicPN thus increases the distance between the detectable label presenton the sicPN and the surface to which the polynucleotide was templated.This increase in distance allows detection of the previously quencheddetectable label, and indicates the presence of the targetpolynucleotide.

Thus, in one embodiment a method is provided in which a plurality ofpolynucleotides are used to produce a nanoconjugate by a methoddescribed herein. The polynucleotides are designed to be able tohybridize to one or more target polynucleotides under stringentconditions. Hybridization can be performed under different stringencyconditions known in the art and as discussed herein. Followingproduction of a nanoconjugate with the plurality of polynucleotides, aplurality of sicPNs optionally comprising a detectable label is addedand allowed to hybridize with the polynucleotides that are part of thenanoconjugate. In some aspects, the plurality of polynucleotides and thesicPNs are first hybridized to each other, and then duplexes used toproduce the nanoconjugate. Regardless of the order in which theplurality of polynucleotide is hybridized to the plurality of sicPNs andthe duplex is used to produce the nanoconjugate, the next step is tocontact the nanoconjugate with a target polynucleotide. The targetpolynucleotide can, in various aspects, be in a solution, or it can beinside a cell. It will be understood that in some aspects, the solutionis being tested for the presence or absence of the target polynucleotidewhile in other aspects, the solution is being tested for the relativeamount of the target polynucleotide.

After contacting the duplex with the target polynucleotide, the targetpolynucleotide will displace and/or release the sicPN as a result of itshybridization with the polynucleotide that is part of the nanoconjugate.The displacement and release of the sicPN allows an increase in distancebetween the surface and the sicPN, thus resulting in the label on thesicPN being rendered detectable. The amount of label that is detected asa result of displacement and release of the sicPN is related to theamount of the target polynucleotide present in the solution. In general,an increase in the amount of detectable label correlates with anincrease in the number of target polynucleotides in the solution.

In some embodiments it is desirable to detect more than one targetpolynucleotide in a solution. In these embodiments, more than one sicPNis used, and each sicPN comprises a unique detectable label.Accordingly, each target polynucleotide, as well as its relative amount,is individually detectable based on the detection of each uniquedetectable label.

In some embodiments, the compositions of the disclosure are useful innano-flare technology. The nano-flare has been previously described inthe context of polynucleotide-functionalized nanoparticles that can takeadvantage of a sicPN architecture for fluorescent detection ofbiomolecule levels inside a living cell [described in WO 2008/098248,incorporated by reference herein in its entirety]. In this system thesicPN acts as the “flare” and is detectably labeled and displaced orreleased from the surface by an incoming target polynucleotide. It isthus contemplated that the nano-flare technology is useful in thecontext of the nanoconjugates described herein.

In further aspects, the nanoconjugate is used to detect the presence oramount of cysteine in a sample, comprising providing a first mixturecomprising a complex comprising Hg2+ and a population of nanoconjugates,wherein the population comprises nanoconjugates comprising one of a pairof single stranded polynucleotides and nanoconjugates comprising theother single stranded polynucleotide of the pair, wherein the pair formsa double stranded duplex under appropriate conditions having at leastone nucleotide mismatch, contacting the first mixture with a samplesuspected of having cysteine to form a second mixture, and detecting themelting point of the double stranded duplex in the second mixture,wherein the melting point is indicative of the presence or amount ofcysteine in the sample. In some aspects, the nucleotide mismatch is aninternal nucleotide mismatch. In a further aspect, the mismatch is a T-Tmismatch. In still a further aspect, the sample comprising cysteine hasa melting point at least about 5° C. lower than a sample withoutcysteine.

Methods of Inhibiting Gene Expression

Additional methods provided by the disclosure include methods ofinhibiting expression of a gene product expressed from a targetpolynucleotide comprising contacting the target polynucleotide with acomposition as described herein, wherein the contacting is sufficient toinhibit expression of the gene product. Inhibition of the gene productresults from the hybridization of a target polynucleotide with acomposition of the disclosure.

It is understood in the art that the sequence of a polynucleotide thatis part of a nanoconjugate need not be 100% complementary to that of itstarget polynucleotide in order to specifically hybridize to the targetpolynucleotide. Moreover, a polynucleotide that is part of ananoconojugate may hybridize to a target polynucleotide over one or moresegments such that intervening or adjacent segments are not involved inthe hybridization event (for example and without limitation, a loopstructure or hairpin structure). The percent complementarity isdetermined over the length of the polynucleotide that is part of thenanoconjugate. For example, given a nanoconjugate comprising apolynucleotide in which 18 of 20 nucleotides of the polynucleotide arecomplementary to a 20 nucleotide region in a target polynucleotide of100 nucleotides total length, the polynucleotide that is part of thenanoconjugate would be 90 percent complementary. In this example, theremaining noncomplementary nucleotides may be clustered or interspersedwith complementary nucleotides and need not be contiguous to each otheror to complementary nucleotides. Percent complementarity of apolynucleotide that is part of a nanoconjugate with a region of a targetpolynucleotide can be determined routinely using BLAST programs (basiclocal alignment search tools) and PowerBLAST programs known in the art(Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden,Genome Res., 1997, 7, 649-656).

Methods for inhibiting gene product expression provided include thosewherein expression of the target gene product is inhibited by at leastabout 5%, at least about 10%, at least about 15%, at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, at least about 99%, or 100% compared to gene product expression inthe absence of a nanoconjugate comprising a biomolecule and/ornon-biomolecule. In other words, methods provided embrace those whichresults in essentially any degree of inhibition of expression of atarget gene product.

The degree of inhibition is determined in vivo from a body fluid sampleor from a biopsy sample or by imaging techniques well known in the art.Alternatively, the degree of inhibition is determined in vitro in a cellculture assay, generally as a predictable measure of a degree ofinhibition that can be expected in vivo resulting from use of acomposition as described herein. It is contemplated by the disclosurethat the inhibition of a target polynucleotide is used to assess theeffects of the inhibition on a given cell. By way of non-limitingexamples, one can study the effect of the inhibition of a gene productwherein the gene product is part of a signal transduction pathway.Alternatively, one can study the inhibition of a gene product whereinthe gene product is hypothesized to be involved in an apoptotic pathway.

It will be understood that any of the methods described herein can beused in combination to achieve a desired result. For example and withoutlimitation, methods described herein can be combined to allow one toboth detect a target polynucleotide as well as regulate its expression.In some embodiments, this combination can be used to quantitate theinhibition of target polynucleotide expression over time either in vitroor in vivo. The quantitation over time is achieved, in one aspect, byremoving cells from a culture at specified time points and assessing therelative level of expression of a target polynucleotide at each timepoint. A decrease in the amount of target polynucleotide as assessed, inone aspect, through visualization of a detectable label, over timeindicates the rate of inhibition of the target polynucleotide.

Thus, determining the effectiveness of a given polynucleotide tohybridize to and inhibit the expression of a target polynucleotide, aswell as determining the effect of inhibition of a given polynucleotideon a cell, are aspects that are contemplated.

Imaging Methods Magnetic Resonance Imaging (MRI)

In certain embodiments, the MRI contrast agent conjugated to apolynucleotide is iron or paramagnetic radiotracers and/or complexes,including but not limited to gadolinium, xenon, iron oxide, and copper.

Fluorescence

Methods are provided wherein presence of a composition of the disclosureis detected by an observable change. In one aspect, presence of thecomposition gives rise to a color change which is observed with a devicecapable of detecting a specific marker as disclosed herein. For exampleand without limitation, a fluorescence microscope can detect thepresence of a fluorophore that is conjugated to a polynucleotide, whichis part of a nanoconjugate.

Complex Visualization through Catalytic Metal Deposition

Methods described herein include depositing a metal on a complex formedbetween a nanoconjugate as defined herein and a target molecule toenhance detection of the complex. Metal is deposited on thenanoparticle/target molecule when the nanoparticle/target moleculecomplex is contacted with a metal enhancing solution under conditionsthat cause a layer of the metal to deposit on the complex. Thus, thepresent disclosure also provides a composition comprising ananoconjugate, the nanoconjugate having a single catalytic metaldeposit, the composition having an average diameter of at least about250 nanometers. In some embodiments, the average diameter is from about250 nanometers to about 5000 nanometers. In some aspects, more than onecatalytic metal deposit is contemplated.

A metal enhancing solution, as used herein, is a solution that iscontacted with a nanoconjugate-target molecule complex to deposit ametal on the complex. In various aspects and depending on the type ofmetal being deposited, the metal enhancing solution comprises, forexample and without limitation, HAuCl₄, silver nitrate, NH₂OH andhydroquinone.

In some embodiments, the target molecule is immobilized on a supportwhen it is contacted with the nanoconjugate. A support, as used herein,includes but is not limited to a column, a membrane, or a glass orplastic surface. A glass surface support includes but is not limited toa bead or a slide. Plastic surfaces contemplated by the presentdisclosure include but are not limited to slides, and microtiter plates.Microarrays are additional supports contemplated by the presentdisclosure, and are typically either glass, silicon-based or a polymer.Microarrays are known to those of ordinary skill in the art and comprisetarget molecules arranged on the support in addressable locations.Microarrays can be purchased from, for example and without limitation,Affymetrix, Inc.

In some embodiments, the target molecule is in a solution. In this typeof assay, a nanoconjugate is contacted with the target molecule in asolution to form a nanoparticle/target molecule complex that is thendetected following deposition of a metal on the complex. Methods of thistype are useful whether the target molecule is in a solution or in abody fluid. For example and without limitation, a solution as usedherein means a buffered solution, water, or an organic solution. Bodyfluids include without limitation blood (serum or plasma), lymphaticfluid, cerebrospinal fluid, semen, urine, synovial fluid, tears, mucous,and saliva and can be obtained by methods routine to those skilled inthe art.

The disclosure also contemplates the use of the compositions and methodsdescribed herein for detecting a metal ion (for example and withoutlimitation, mercuric ion (Hg²⁺)). In these aspects, the method takesadvantage of the cooperative binding and catalytic properties of thenanoconjugates comprising a DNA polynucleotide and the selective bindingof a thyminethymine mismatch for Hg²⁺ [Lee et al., Anal. Chem. 80:6805-6808 (2008)].

Methods described herein are also contemplated for use in combinationwith the biobarcode assay. The biobarcode assay is generally describedin U.S. Pat. Nos. 6,974,669 and 7,323,309, each of which is incorporatedherein by reference in its entirety.

Methods of the disclosure include those wherein silver or gold orcombinations thereof are deposited on a nanoconjugate in a complex witha target molecule.

In one embodiment, methods of silver deposition on a nanoconjugate asdescribed herein yield a limit of detection of a target molecule ofabout 3 pM after a single silver deposition. In another aspect, a secondsilver deposition improves the limit of detection to about 30 fM. Thus,the number of depositions of silver relates to the limit of detection ofa target molecule. Accordingly, one of ordinary skill in the art willunderstand that the methods of the present disclosure may be tailored tocorrelate with a given concentration of target molecule. For example andwithout limitation, for a target molecule concentration of 30 fM, twosilver depositions can be used. Concentrations of target moleculesuitable for detection by silver deposition are about 3 pM, about 2 pM,about 1 pM, about 0.5 pM, about 400 fM, about 300 fM, about 200 fM,about 100 fM or less.

In methods provided, a nanoconjugate is contacted with a samplecomprising a first molecule under conditions that allow complexformation between the nanoconjugate and the first molecule.

Methods are also provided wherein a second molecule is contacted withthe first molecule under conditions that allow complex formation priorto the contacting of the nanoconjugate with the first molecule.

Method are also contemplated wherein a target molecule is attached to asecond nanoconjugate that associates with the first nanoconjugate. Insome aspects, the second nanoconjugate is immobilized on a solidsupport. In other aspects, the second nanoconjugate is in a solution.

Methods provided also generally contemplate contacting a compositioncomprising a nanoconjugate with more than one target molecules.Accordingly, in some aspects it is contemplated that a nanoconjugatecomprising more than one polypeptide and/or polynucleotide, is able tosimultaneously recognize and associate with more than one targetmolecule.

In further embodiments, a target polynucleotide is identified using a“sandwich” protocol for high-throughput detection and identification.For example and without limitation, a polynucleotide that recognizes andselectively associates with the target polynucleotide is immobilized ona solid support. The sample comprising the target polynucleotide iscontacted with the solid support comprising the polynucleotide, thusallowing an association to occur. Following removal of non-specificinteractions, a composition comprising a nanoconjugate as describedherein is added. In these aspects, the nanoconjugate comprises amolecule that selectively associates with the target polynucleotide,thus generating the “sandwich” of polynucleotide-targetpolynucleotide-nanoconjugate. This complex is then exposed to a metaldeposition process as described herein, resulting in highly sensitivedetection. Quantification of the interaction allows for determinationsrelating but not limited to disease progression, therapeuticeffectiveness, disease identification, and disease susceptibility.

Additional description of catalytic deposition of metal on a complexformed between a nanoconjugate as defined herein and a target moleculeto enhance detection of the complex is found in U.S. application Ser.No. 12/770,488, which is incorporated by reference herein in itsentirety.

Detecting Modulation of Transcription of a Target Polynucleotide

Methods provided by the disclosure include a method of detectingmodulation of transcription of a target polynucleotide comprisingadministering a nanoconjugate and a transcriptional regulator andmeasuring a detectable change, wherein the transcriptional regulatorincreases or decreases transcription of the target polynucleotide in atarget cell relative to a transcription level in the absence of thetranscriptional regulator.

The disclosure also contemplates methods to identify the targetpolynucleotide. In some aspects of these methods, a library ofpolynucleotides is screened for its ability to detect the increase ordecrease in transcription of the target polynucleotide. The library, invarious aspects, is a polynucleotide library. In some aspects of thesemethods, a double stranded polynucleotide comprising a known sequence isused to produce a nanoconjugate, creating a first nanoconjugate. In someaspects, one strand of the double stranded polynucleotide furthercomprises a detectable marker that is quenched while the two strands ofthe polynucleotide remain hybridized to each other. The nanoconjugate isthen contacted with a target cell concurrently with a transcriptionalregulator. If the polynucleotide of known sequence that is used toproduce the nanoconjugate hybridizes with the target polynucleotide, itresults in a detectable change. The detectable change, in some aspects,is fluorescence. Observation of a detectable change that issignificantly different from the detectable change observed bycontacting the target cell with a second nanoconjugate in which thepolynucleotide comprises a different sequence than the firstnanoconjugate is indicative of identifying the target polynucleotide.Thus, in further aspects, each nanoconjugate comprises a polynucleotideof known sequence, and in still further aspects, an increase or decreasein the detectable change when the transcriptional regulator isadministered relative to the detectable change measured when a differentnanoparticle comprising a polynucleotide within the library isadministered is indicative of identifying the target polynucleotide.Accordingly, in some aspects the methods provide for the identificationof a mRNA that is regulated by a given transcriptional regulator. Invarious aspects, the mRNA is increased, and in some aspects the mRNA isdecreased.

Local delivery of a composition comprising a nanoconjugate to a human iscontemplated in some aspects of the disclosure. Local delivery involvesthe use of an embolic agent in combination with interventional radiologyand a composition of the disclosure.

Use of a Nanoconjugate as a Probe

The nanoconjugates are, in one aspect, used as probes in diagnosticassays for detecting nucleic acids.

Some embodiments of the method of detecting a target nucleic acidutilize a substrate. Any substrate can be used which allows observationof the detectable change. Suitable substrates include transparent solidsurfaces (e.g., glass, quartz, plastics and other polymers), opaquesolid surface (e.g., white solid surfaces, such as TLC silica plates,filter paper, glass fiber filters, cellulose nitrate membranes, nylonmembranes), and conducting solid surfaces (e.g., indium-tin-oxide(ITO)). The substrate can be any shape or thickness, but generally willbe flat and thin. Preferred are transparent substrates such as glass(e.g., glass slides) or plastics (e.g., wells of microtiter plates).Methods of attaching polynucleotides to a substrate and uses thereofwith respect to nanoconjugates are disclosed in U.S. Patent Application20020172953, incorporated herein by reference in its entirety.

By employing a substrate, the detectable change can be amplified and thesensitivity of the assay increased. In one aspect, the method comprisesthe steps of contacting a target polynucleotide with a substrate havinga polynucleotide attached thereto, the polynucleotide (i) having asequence complementary to a first portion of the sequence of the targetnucleic acid, the contacting step performed under conditions effectiveto allow hybridization of the polynucleotide on the substrate with thetarget nucleic acid, and (ii) contacting the target nucleic acid boundto the substrate with a first type of nanoconjugate having apolynucleotide attached thereto, the polynucleotide having a sequencecomplementary to a second portion of the sequence of the target nucleicacid, the contacting step performed under conditions effective to allowhybridization of the polynucleotide that is part of the nanoconjugatewith the target nucleic acid. Next, the first type of nanoconjugatebound to the substrate is contacted with a second type of nanoconjugatecomprising a polynucleotide, the polynucleotide on the second type ofnanoconjugate having a sequence complementary to at least a portion ofthe sequence of the polynucleotide used to produce the first type ofnanoconjugate, the contacting step taking place under conditionseffective to allow hybridization of the polynucleotides on the first andsecond types of nanoconjugates. Finally, a detectable change produced bythese hybridizations is observed.

The detectable change that occurs upon hybridization of thepolynucleotides on the nanoconjugates to the nucleic acid may be a colorchange, the formation of aggregates of the nanoconjugates, or theprecipitation of the aggregated nanoconjugates. The color changes can beobserved with the naked eye or spectroscopically. The formation ofaggregates of the nanoconjugates can be observed by electron microscopyor by nephelometry. The precipitation of the aggregated nanoconjugatescan be observed with the naked eye or microscopically. Preferred arechanges observable with the naked eye. Particularly preferred is a colorchange observable with the naked eye.

The methods of detecting target nucleic acid hybridization based onobserving a color change with the naked eye are cheap, fast, simple,robust (the reagents are stable), do not require specialized orexpensive equipment, and little or no instrumentation is required. Theseadvantages make them particularly suitable for use in, e.g., researchand analytical laboratories in DNA sequencing, in the field to detectthe presence of specific pathogens, in the doctor's office for quickidentification of an infection to assist in prescribing a drug fortreatment, and in homes and health centers for inexpensive first-linescreening.

A nanoconjugate comprising a polynucleotide can be used in an assay totarget a target molecule of interest. Thus, the nanoconjugate comprisinga polynucleotide can be used in an assay such as a bio barcode assay.See, e.g., U.S. Pat. Nos. 6,361,944; 6,417,340; 6,495,324; 6,506,564;6,582,921; 6,602,669; 6,610,491; 6,678,548; 6,677,122; 6682,895;6,709,825; 6,720,147; 6,720,411; 6,750,016; 6,759,199; 6,767,702;6,773,884; 6,777,186; 6,812,334; 6,818,753; 6,828,432; 6,827,979;6,861,221; and 6,878,814, the disclosures of which are incorporatedherein by reference.

Dosing And Pharmaceutical Compositions

It will be appreciated that any of the compositions described herein maybe administered to a mammal in a therapeutically effective amount toachieve a desired therapeutic effect.

The term “therapeutically effective amount”, as used herein, refers toan amount of a composition sufficient to treat, ameliorate, or preventthe identified disease or condition, or to exhibit a detectabletherapeutic, prophylactic, or inhibitory effect. The effect can bedetected by, for example, an improvement in clinical condition,reduction in symptoms, or by an assay described herein. The preciseeffective amount for a subject will depend upon the subject's bodyweight, size, and health; the nature and extent of the condition; andthe antibiotic composition or combination of compositions selected foradministration. Therapeutically effective amounts for a given situationcan be determined by routine experimentation that is within the skilland judgment of the clinician.

The compositions described herein may be formulated in pharmaceuticalcompositions with a pharmaceutically acceptable excipient, carrier, ordiluent. The compound or composition can be administered by any routethat permits treatment of, for example and without limitation, adisease, disorder or infection as described herein. A preferred route ofadministration is oral administration. Additionally, the compound orcomposition comprising the antibiotic composition may be delivered to apatient using any standard route of administration, includingparenterally, such as intravenously, intraperitoneally, intrapulmonary,subcutaneously or intramuscularly, intrathecally, transdermally (asdescribed herein), rectally, orally, nasally or by inhalation.

Slow release formulations may also be prepared from the agents describedherein in order to achieve a controlled release of the active agent incontact with the body fluids in the gastro intestinal tract, and toprovide a substantial constant and effective level of the active agentin the blood plasma. The crystal form may be embedded for this purposein a polymer matrix of a biological degradable polymer, a water-solublepolymer or a mixture of both, and optionally suitable surfactants.Embedding can mean in this context the incorporation of micro-particlesin a matrix of polymers. Controlled release formulations are alsoobtained through encapsulation of dispersed micro-particles oremulsified micro-droplets via known dispersion or emulsion coatingtechnologies.

Administration may take the form of single dose administration, or thecompound of the embodiments can be administered over a period of time,either in divided doses or in a continuous-release formulation oradministration method (e.g., a pump). However the compounds of theembodiments are administered to the subject, the amounts of compoundadministered and the route of administration chosen should be selectedto permit efficacious treatment of the disease condition. Administrationof combinations of therapeutic agents (i.e., combination therapy) isalso contemplated, provided at least one of the therapeutic agents is inassociation with a nanoconjugate as described herein.

In an embodiment, the pharmaceutical compositions may be formulated withpharmaceutically acceptable excipients such as carriers, solvents,stabilizers, adjuvants, diluents, etc., depending upon the particularmode of administration and dosage form. The pharmaceutical compositionsshould generally be formulated to achieve a physiologically compatiblepH, and may range from a pH of about 3 to a pH of about 11, preferablyabout pH 3 to about pH 7, depending on the formulation and route ofadministration. In alternative embodiments, it may be preferred that thepH is adjusted to a range from about pH 5.0 to about pH 8. Moreparticularly, the pharmaceutical compositions comprises in variousaspects a therapeutically or prophylactically effective amount of atleast one composition as described herein, together with one or morepharmaceutically acceptable excipients. As described herein, thepharmaceutical compositions may optionally comprise a combination of thecompounds described herein.

The term “pharmaceutically acceptable excipient” refers to an excipientfor administration of a pharmaceutical agent, such as the compoundsdescribed herein. The term refers to any pharmaceutical excipient thatmay be administered without undue toxicity.

Pharmaceutically acceptable excipients are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there exists awide variety of suitable formulations of pharmaceutical compositions(see, e.g., Remington's Pharmaceutical Sciences).

Suitable excipients may be carrier molecules that include large, slowlymetabolized macromolecules such as proteins, polysaccharides, polylacticacids, polyglycolic acids, polymeric amino acids, amino acid copolymers,and inactive virus particles. Other exemplary excipients includeantioxidants (e.g., ascorbic acid), chelating agents (e.g., EDTA),carbohydrates (e.g., dextrin, hydroxyalkylcellulose, and/orhydroxyalkylmethylcellulose), stearic acid, liquids (e.g., oils, water,saline, glycerol and/or ethanol) wetting or emulsifying agents, pHbuffering substances, and the like. Liposomes are also included withinthe definition of pharmaceutically acceptable excipients.

Additionally, the pharmaceutical compositions may be in the form of asterile injectable preparation, such as a sterile injectable aqueousemulsion or oleaginous suspension. This emulsion or suspension may beformulated by a person of ordinary skill in the art using suitabledispersing or wetting agents and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent,such as a solution in 1,2-propane-diol.

The sterile injectable preparation may also be prepared as a lyophilizedpowder. In addition, sterile fixed oils may be employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employedincluding synthetic mono- or diglycerides. In addition, fatty acids(e.g., oleic acid) may likewise be used in the preparation ofinjectables.

Transdermal Delivery

In some aspects of the disclosure, a method of dermal delivery of ananoconjugate is provided comprising the step of administering acomposition comprising the nanoconjugate and a dermal vehicle to theskin of a patient in need thereof.

In one aspect, the delivery of the nanoconjugate is transdermal. Inanother aspect, the delivery of the nanoconjugate is topical. In anotheraspect, the delivery of the nanoconjugate is to the epidermis and dermisafter topical application. In some embodiments, the dermal vehiclecomprises an ointment. In some aspects, the ointment is Aquaphor.

In further embodiments of the methods, the administration of thecomposition ameliorates a skin disorder. In various embodiments, theskin disorder is selected from the group consisting of cancer, a geneticdisorder, aging, inflammation, infection, and cosmetic disfigurement.

See PCT/US2010/27363, incorporated by reference herein in its entirety,for further description of dermal delivery of nanoparticle compositionsand methods of their use.

Vehicles

In some embodiments, compositions and methods of the present disclosurecomprise vehicles. As used herein, a “vehicle” is a base compound withwhich an nanoconjugate composition is associated.

Vehicles useful in the compositions and methods of the presentdisclosure are known to those of ordinary skill in the art and includewithout limitation an ointment, cream, lotion, gel, foam, buffersolution (for example and without limitation, Ringer's solution andisotonic sodium chloride solution) or water. In some embodiments,vehicles comprise one or more additional substances including but notlimited to salicylic acid, alpha-hydroxy acids, or urea that enhance thepenetration through the stratum corneum.

In various aspects, vehicles contemplated for use in the compositionsand methods of the present disclosure include, but are not limited to,Aquaphor® healing ointment, A+D, polyethylene glycol (PEG), glycerol,mineral oil, Vaseline Intensive Care cream (comprising mineral oil andglycerin), petroleum jelly, DML (comprising petrolatum, glycerin and PEG20), DML (comprising petrolatum, glycerin and PEG 100), Eucerinmoisturizing cream, Cetaphil (comprising petrolatum, glycerol and PEG30), Cetaphil, CeraVe (comprising petrolatum and glycerin), CeraVe(comprising glycerin, EDTA and cholesterol), Jergens (comprisingpetrolatum, glycerin and mineral oil), and Nivea (comprising petrolatum,glycerin and mineral oil). One of ordinary skill in the art willunderstand from the above list that additional vehicles are useful inthe compositions and methods of the present disclosure.

An ointment, as used herein, is a formulation of water in oil. A creamas used herein is a formulation of oil in water. In general, a lotionhas more water than a cream or an ointment; a gel comprises alcohol, anda foam is a substance that is formed by trapping gas bubbles in aliquid. These terms are understood by those of ordinary skill in theart.

Embolic Agents

Administration of an embolic agent in combination with a composition ofthe disclosure is also contemplated. Embolic agents serve to increaselocalized drug concentration in target sites through selective occlusionof blood vessels by purposely introducing emboli, while decreasing drugwashout by decreasing arterial inflow. Thus, a composition comprising ananoparticle comprising a polynucleotide, wherein the polynucleotide isconjugated to a contrast agent through a conjugation site would remainat a target site for a longer period of time in combination with anembolic agent relative to the period of time the composition wouldremain at the target site without the embolic agent. Accordingly, insome embodiments, the present disclosure contemplates the use of acomposition as described herein in combination with an embolic agent.

In various aspects of the compositions and methods of the disclosure,the embolic agent to be used is selected from the group consisting of alipid emulsion (for example and without limitation, ethiodized oil orlipiodol), gelatin sponge, tris acetyl gelatin microspheres,embolization coils, ethanol, small molecule drugs, biodegradablemicrospheres, non-biodegradable microspheres or polymers, andself-assembling embolic material.

The compositions disclosed herein are administered by any route thatpermits imaging of the tissue or cell that is desired, and/or treatmentof the disease or condition. In one aspect the route of administrationis intraarterial administration. Additionally, the compositioncomprising a nanoconjugate is delivered to a patient using any standardroute of administration, including but not limited to orally,parenterally, such as intravenously, intraperitoneally, intrapulmonary,intracardiac, intraosseous infusion (“IO”), subcutaneously orintramuscularly, intrathecally, transdermally, intradermally, rectally,orally, nasally or by inhalation or transmucosal delivery. Directinjection of a composition provided herein is also contemplated and, insome aspects, is delivered via a hypodermic needle. Slow releaseformulations may also be prepared from the compositions described hereinin order to achieve a controlled release of one or more components of acomposition as described herein in contact with the body fluids and toprovide a substantially constant and effective level of one or morecomponents of a composition in the blood plasma.

Target Site Identification and Composition Delivery

Provided herein are methods of delivering a contrast agent to a cellcomprising contacting the cell with a composition of the disclosureunder conditions sufficient to deliver the contrast agent to the cell.Following delivery of the composition, in some aspects the methodfurther comprises the step of detecting the contrast agent. Detectingthe contrast agent is performed by any of the methods known in the art,including those described herein.

In a specific embodiment, the contrast agent is detected using animaging procedure, and in various aspects, the imaging procedure isselected from the group consisting of MRI, CT, and fluorescence.

Methods provided also include those wherein a composition of thedisclosure is locally delivered to a target site. Once the target sitehas been identified, a composition of the disclosure is delivered, inone aspect, intraarterially. In another aspect, a composition of thedisclosure is delivered intravenously. Target cells for delivery of acomposition of the disclosure are, in various aspects, selected from thegroup consisting of a cancer cell, a stem cell, a T-cell, and a β-isletcell.

In various aspects, the target site is a site of pathogenesis.

In some aspects, the site of pathogenesis is cancer. In various aspects,the cancer is selected from the group consisting of liver, pancreatic,stomach, colorectal, prostate, testicular, renal cell, breast, bladder,ureteral, brain, lung, connective tissue, hematological, cardiovascular,lymphatic, skin, bone, eye, nasopharyngeal, laryngeal, esophagus, oralmembrane, tongue, thyroid, parotid, mediastinum, ovary, uterus, adnexal,small bowel, appendix, carcinoid, gall bladder, pituitary, cancerarising from metastatic spread, and cancer arising from endodermal,mesodermal or ectodermally-derived tissues.

In some embodiments, the site of pathogenesis is a solid organ disease.In various aspects, the solid organ is selected from the groupconsisting of heart, liver, pancreas, prostate, brain, eye, thyroid,pituitary, parotid, skin, spleen, stomach, esophagus, gall bladder,small bowel, bile duct, appendix, colon, rectum, breast, bladder,kidney, ureter, lung, and a endodermally-, ectodermally- ormesodermally-derived tissues.

Activation of a Chemotherapeutic Agent

According to the disclosure, it is contemplated that a chemotherapeuticagent that is attached to a nanoconjugate as described herein isactivated upon entry into a cell. In some aspects, the activatedchemotherapeutic agent confers an increase in cytotoxicity relative to achemotherapeutic agent that is not attached to a polynucleotide, whereinthe polynucleotide is part of a nanoconjugate, and wherein the increasein cytotoxicity is measured using an in vitro cell culture assay. The invitro cell culture assay is, for example and without limitation, a(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT)assay. Accordingly, the increase in cytotoxicity described above iscoupled with the reduced toxicity of the chemotherapeutic agent which isattached to a polynucleotide that is part of a nanoconjugate prior toits entry into a cell.

Target Molecules

It is contemplated by the disclosure that any of the compositionsdescribed herein can be used to detect a target molecule. In variousaspects, the target molecule is a polynucleotide, and the polynucleotideis either eukaryotic, prokaryotic, or viral.

In some aspects, the target molecule is a polynucleotide.

If a polynucleotide is present in small amounts, it may be amplified bymethods known in the art. See, e.g., Sambrook et al., Molecular Cloning:A Laboratory Manual (2nd ed. 1989) and B. D. Hames and S. J. Higgins,Eds., Gene Probes 1 (IRL Press, New York, 1995). Generally, but withoutlimitation, polymerase chain reaction (PCR) amplification can beperformed to increase the concentration of a target nucleic acid to adegree that it can be more easily detected.

In various embodiments, methods provided include those wherein thetarget polynucleotide is a mRNA encoding a gene product and translationof the gene product is inhibited, or the target polynucleotide is DNA ina gene encoding a gene product and transcription of the gene product isinhibited. In methods wherein the target polynucleotide is DNA, thepolynucleotide is in certain aspects DNA which encodes the gene productbeing inhibited. In other methods, the DNA is complementary to a codingregion for the gene product. In still other aspects, the DNA encodes aregulatory element necessary for expression of the gene product.“Regulatory elements” include, but are not limited to enhancers,promoters, silencers, polyadenylation signals, regulatory proteinbinding elements, regulatory introns, ribosome entry sites, and thelike. In still another aspect, the target polynucleotide is a sequencewhich is required for endogenous replication. In further embodiments,the target molecule is a microRNA (miRNA).

Anti-Prokaryotic Target Polynucleotides

For prokaryotic target polynucleotides, in various aspects, thepolynucleotide is genomic DNA or RNA transcribed from genomic DNA. Foreukaryotic target polynucleotides, the polynucleotide is an animalpolynucleotide, a plant polynucleotide, a fungal polynucleotide,including yeast polynucleotides. As above, the target polynucleotide iseither a genomic DNA or RNA transcribed from a genomic DNA sequence. Incertain aspects, the target polynucleotide is a mitochondrialpolynucleotide. For viral target polynucleotides, the polynucleotide isviral genomic RNA, viral genomic DNA, or RNA transcribed from viralgenomic DNA.

In one embodiment, the polynucleotides of the invention are designed tohybridize to a target prokaryotic sequence under physiologicalconditions.

It will be understood that one of skill in the art may readily determineappropriate targets for nanoconjugates comprising a polynucleotide, anddesign and synthesize polynucleotides using techniques known in the art.Targets can be identified by obtaining, e.g., the sequence of a targetnucleic acid of interest (e.g. from GenBank) and aligning it with othernucleic acid sequences using, for example, the MacVector 6.0 program, aClustalW algorithm, the BLOSUM 30 matrix, and default parameters, whichinclude an open gap penalty of 10 and an extended gap penalty of 5.0 fornucleic acid alignments.

Any essential prokaryotic gene is contemplated as a target gene usingthe methods of the present disclosure. As described above, an essentialprokaryotic gene for any prokaryotic species can be determined using avariety of methods including those described by Gerdes for E. coli[Gerdes et al., J Bacteriol. 185(19): 5673-84, 2003]. Many essentialgenes are conserved across the bacterial kingdom thereby providingadditional guidance in target selection. Target gene sequences can beidentified using readily available bioinformatics resources such asthose maintained by the National Center for Biotechnology Information(NCBI).

Nanoconjugates comprising a polynucleotide showing optimal activity arethen tested in animal models, or veterinary animals, prior to use fortreating human infection.

Target Sequences for Cell-Division and Cell-Cycle Target Proteins

The polynucleotides of the present disclosure are designed to hybridizeto a sequence of a prokaryotic nucleic acid that encodes an essentialprokaryotic gene. Exemplary genes include but are not limited to thoserequired for cell division, cell cycle proteins, or genes required forlipid biosynthesis or nucleic acid replication.

For each of these three proteins, Table 1 of U.S. Patent ApplicationNumber 20080194463, incorporated by reference herein in its entirety,provides exemplary bacterial sequences which contain a target sequencefor each of a number of important pathogenic bacteria. The genesequences are derived from the GenBank Reference full genome sequencefor each bacterial strain.

Target Sequences for Prokaryotic 16S Ribosomal RNA

In one embodiment, the polynucleotides of the invention are designed tohybridize to a sequence encoding a bacterial 16S rRNA nucleic acidsequence under physiological conditions, with a T_(m) substantiallygreater than 37° C., e.g., at least 45° C. and preferably 60° C.-80° C.

Exemplary bacteria and associated GenBank Accession Nos. for 16S rRNAsequences are provided in Table 1 of U.S. Pat. No. 6,677,153,incorporated by reference herein in its entirety.

Additional Target Molecules

The target molecule may be in cells, tissue samples, or biologicalfluids, as also known in the art.

In various embodiments the disclosure contemplates that more than onetarget polynucleotide is detected in the target cell.

In further embodiments the target molecule is an ion. The presentdisclosure contemplates that in one aspect the ion is nitrite (NO2-). Insome aspects, the ion is a metal ion that is selected from the groupconsisting of mercury (Hg2+), Cu2+ and UO2+.

Kits

Also provided are kits comprising a composition of the disclosure. Inone embodiment, the kit comprises at least one container, the containerholding at least one type of nanoconjugate as described hereincomprising one or more polynucleotides as described herein. Thepolynucleotides that are part of the first type of nanoconjugate haveone or more sequences complementary (or sufficiently complementary asdisclosed herein) to one or more sequences of a first portion of atarget polynucleotide. The container optionally includes one or moreadditional type of nanoconjugates comprising a polynucleotide with asequence complementary to one or more sequence of a second portion ofthe target polynucleotide.

In another embodiment, the kit comprises at least two containers. Thefirst container holds one or more nanoconjugates as disclosed hereincomprising one or more biomolecules and/or non-biomolecules as describedherein which can associate with one or more portions of a targetbiomolecule and/or non-biomolecule. The second container holds one ormore nanoconjugates comprising one or more biomolecules and/ornon-biomolecules can associate with one or more sequences of the same ora different portion of the target biomolecule and/or non-biomolecule.

In another embodiment, the kits have biomolecules and/ornon-biomolecules and nanoparticles in separate containers, and thenanoconjugates are produced prior to use for a method described herein.In one aspect, the biomolecules and/or non-biomolecules and/or thenanoparticles are functionalized so that the nanoconjugates can beproduced. Alternatively, the biomolecules and/or non-biomolecules and/ornanoparticles are provided in the kit without functional groups, inwhich case they must be functionalized prior to performing the assay. Inadditional aspects, a chemical is provided that facilitates thecrosslinking of the biomolecules and/or non-biomolecules.

In various aspects of the kits provided, biomolecules and/ornon-biomolecules include a label or the kit includes a label which canbe attached to the biomolecules and/or non-biomolecules. Alternatively,the kits include labeled nanoparticles or labels which can be attachedto the nanoparticles. In each embodiment, the kit optionally includesinstructions, each container contains a label, the kit itself includes alabel, the kit optionally includes one or more non-specific biomolecules(for use as controls).

EXAMPLES Example 1 Materials

All materials were purchased from Sigma-Aldrich and used without furtherpurification, unless otherwise indicated. TEM characterization wasconducted on a Hitachi H8100 electron microscope. NMR experiments wereperformed using a Bruker Avance III 500 MHz coupled with a DCHCryoProbe. DLS data were acquired from a MALVERN Zetasizer, Nano-ZS. IRresults were obtained from a Bruker TENSOR 37, and analyzed using theOPUS software. MALDI-TOF measurements were carried out on a BrukerAutoflex III SmartBeam mass spectrometer.

Synthesis of poly(N-(2-(3-(prop-2-ynyloxy)propanamido)ethyl)acrylamide)1

Polyacrylamidoethylamine120 (PAEAl20) was prepared following literaturereported methods [Zhang et al., Biomaterials 31: 1805 (2010); Zhang etal., Biomaterials 30: 968 (2009)]. PAEAl20 (67.5 mg, 4.9 μmol) wasdissolved in anhydrous DMSO (2 mL), and stirred for 3 hours, before 1 mLDMSO solution containing propargyl-dPEG1-NHS ester (150 mg, 660 μmol,Quanta Biodesign) and diisopropylethylamine (DIPEA, 204 μL 1.17 mmol)was added. The reaction mixture was allowed to stir overnight, dilutedby the addition of DMSO (10 mL), transferred to pre-soaked dialysistubing (MWCO=3.5 kDa), and dialyzed against nanopure water (>18.0 MΩ.cm)for 3 days. The solution was then lyophilized and re-suspended in water(15 mL). A small amount of cloudiness was observed, which was removed byfiltering through a 0.2 μm syringe filter. No residual amine group wasdetected by a ninhydrin test. IR and 1H-13C HSQC NMR spectra of 1 areshown in FIG. 12.

Synthesis of methyl-terminated poly(ethylene glycol)-propargyl etherconjugate 5

Monodisperse mPEG24-amine (39.0 mg, 34.6 μmol, Quanta Biodesign) wasdissolved in 1.0 mL pH=8.0 phosphate buffer, to whichpropargyl-dPEG1-NHS ester (12.1 mg, 53.7 μmol) was added. The mixturewas allowed to be shaken for 12 hours at 4° C. The desired conjugate wasisolated from the reaction mixture by reverse phase HPLC(water/acetonitrile, Varian DYNAMAX C18 column (250×10.0 mm)).MALDI-TOF: 1220.553 [M+Na]+. 1H-13C HSQC NMR: FIG. 11.

Synthesis of 13 nm AuNPs

An aqueous solution of HAuC14 (1 mM, 500 mL) was brought to reflux whilestirring, and then trisodium citrate solution (50 mL, 77.6 mM) was addedquickly to the boiling mixture. The solution was refluxed for anadditional 15 minutes, and allowed to cool to room temperature. Theaverage diameter of the gold nanoparticles was determined by TEM(12.8±1.2 nm). AuNPs of other sizes used in this study were purchasedfrom Ted Pella.

General Method for the Preparation of Nanoconjugates

To 10 mL AuNP solution (10 nM), 10 μL of 10% sodium dodecyl sulfatesolution was added. Then, an aqueous solution containing 1 was added togive a final concentration of 20 nM. The solution was stirred for 2 daysbefore being subjected to centrifugation using an Eppendorf 5424centrifuge at 15,000 rpm for 30 minutes. Supernatant was removed bycareful pipetting, and the AuNP was resuspended in nanopure water. Theprocess was repeated three times to ensure complete removal of excesspolymers. After the final centrifugation, the polymer-coated AuNP wereconcentrated to 1 mL, and 50 μL of 1.0 M KCN aqueous solution was addedto remove the gold core. The resulting solution was then dialyzedagainst nanopure water (>18.0 MΩ.cm) using pre-soaked dialysis tubing(MWCO=6-8 kDa) for 3 days. The final nanoconjugate solution appearedclear and slightly yellow. A large volume of gold nanoparticle templates(>500 mL) was required to prepare sufficient quantity of nanoconjugatesfor NMR and IR analyses.

Example 2

In searching for appropriate orthogonal chemistries that could crosslinka dense monolayer of DNA together on the gold surface, it was discoveredthat poly-alkyne bearing DNA strands autocrosslink on the goldnanoparticle surface without any additional catalysts. In initialexperiments, DNA strands were synthesized that utilized syntheticallymodified bases that could be modified with desired chemical moieties.Because of the modular nature of phosphoramidite chemistry, these basesare incorporated into a polynucleotide sequence at any location. Themodified base that was chosen for this system is an amine-modifiedthymidine residue that can be reacted with an alkyne-NHS ester toproduce an alkyne modified thymidine within the sequence (FIG. 1).However, any moiety that can be converted to an alkyne can be used.Strands were then synthesized that incorporated a thiol moiety forattachment, a crosslinking region (CR) of 10 amine-modified T monomers aspacer of 5 T residues, and a programmable DNA or RNA binding region(BR). The CR was then modified with the alkyne NHS ester, which resultedin a strand with 10 alkyne units in the BR. Two example sequences usedwere 5′ TCA-CTA-TTA-TTTTT-(alkyne-modified T)10-SH 3′ (SEQ ID NO: 1) and5′ TAA-TAG-TGA-TTTTT-(alkyne-modified T)10-SH 3′ (SEQ ID NO: 2).

In a typical experiment, 1 O.D. of DTT-treated alkyne-DNA is added tolmL of 13 nm gold nanoparticles at a concentration of approximately 10nM. Polysorbate 20 (Tween-20) and phosphate buffer (pH 7.4) are thenadded to the nanoparticles for a final concentration of 0.01% Tween-20and 50 mM phosphate buffer. Because the polynucleotides must be as closeas possible for crosslinking, the nanoparticles were brought up to ahigh sodium chloride concentration of 1.0 M to maximize loading. Theparticles were then centrifuged (13.2 k rpm) and resuspended in PBS/SDSthree times to remove excess DNA.

Dissolution of the AuNP core was achieved by using KCN in the presenceof oxygen. When KCN was added to citrate stabilized AuNPs, the color ofthe solution instantly changed from red to purple, resulting from thedestabilization and aggregation of the AuNPs (FIG. 2A). A similar effectis observed for AuNPs densely functionalized with thiolatedpolynucleotides, but the process is slower (FIG. 2A). However, for thealkyne-DNA AuNP, the color slowly changed to a slightly reddish orangecolor during the dissolution process until the solution was clear (FIG.2B). This observation suggested that the alkyne modified DNA formed adense crosslinked shell, which prevented the typical aggregation that istypical of AuNPs being oxidatively dissolved. Furthermore, UV-Visspectra showed a gradual decrease of the plasmon resonance from 524 nmto 518 nm (FIG. 2B), as expected from the decrease of AuNP size (FIG.2C) [Link et al., J. Phys. Chem. B 103(21): 4212-4217 (1999)].

After dialysis, the structures were analyzed by TEM at different stagesof the dissolution process with uranyl acetate staining (FIG. 3). Thedissolution reaction was quenched by rapid spin filtering, which removesall of the KCN and retains the particles on the filter. It was clearthat as the particles dissolve, a dense ligand shell is responsible forthe particles' remarkable stability in KCN. At an intermediate timepoint, staining revealed a dense shell around the particle surface asthe particle shrank in size. After the dissolution process was complete,spherical particles of DNA could clearly be observed.

Example 3

Because these structures were made almost entirely of DNA, gelelectrophoresis was a powerful method to analyze the completeness of thecrosslinking reaction and the quality of the resulting structures. Afterdialysis, the unreacted alkyne-DNA strand was compared with theparticles formed from the templated method. The hollow particlesmigrated much more slowly than the free strands and similarly to theundissolved DNA-AuNP conjugate. Next, the role the density of the DNAplays in the formation of these hollow DNA nanoconjugates was analyzed(FIG. 4). The density of the DNA on the nanoparticle surface couldeasily be controlled with the concentration of sodium ions in theDNA/gold nanoparticle solution during functionalization. At low DNAsurface densities, it was clear that a distribution of crosslinkedproducts was obtained, and with increasing surface density, a dramaticincrease in the size of the crosslinked products was evident. At acritical density obtained from particles salt-aged to 0.5 M NaCl, asharp band appeared at high molecular weight numbers. This band becamethe majority product (approximately 99% by densitometry) from particlesthat were salt-aged to 1.0 M NaCl, which have the maximum surfacedensities.

Example 4

Next, the ability to obtain hollow DNA nanoconjugate from goldnanoparticles of a range of sizes was tested. Indeed, the migration ofthe hollow particles through the gel was directly related to the size ofthe resulting hollow structures, with larger hollow particles migratingslower than the small ones (FIG. 5A). The number of alkynes in the CRwas then varied, while keeping the number of total residues constant, todetermine the minimum alkyne density of this process (by way of example,a strand with 3 alkynes has 7 unmodified T residues in the CR). At athreshold of approximately 5 alkynes in the CR, particles of a similarsize to the ones from the previous experiment are obtained (FIG. 5b ).However, as the number of alkyne units is increased from 5 to largernumbers, the particles migrated slightly faster, which indicates moredensely crosslinked nanoconjugates.

Example 5

After establishing that this method could produce nanoconjugatescomposed entirely of crosslinked DNA, their functional properties wereinvestigated. When polynucleotides are densely arranged on a AuNP'ssurface, many new behaviors emerge including but not limited to elevatedand narrow melting transitions, enhanced binding to targets, reversibledirected assembly, high cellular uptake without transfect agents,dramatic nuclease resistance and robust antisense/RNAi engagement. Theseproperties emerge due to the dense polyvalent arrangement of DNA on thegold nanoparticle's surface. So, if the DNA in the hollow nanoconjugatemaintained their binding ability, the binding properties characteristicof polyvalent DNA would be observable.

To that end, a two nanoparticle system was designed wherein the strandson the nanoparticles were designed such that there is no selfcomplementarity within one sequence, so particles functionalized withone of the strands will be stable in solution. However, when the twoparticles are mixed together, the complementarity of the strands willbring together nanoparticles into a macroscopic polymeric assembly.Because the hollow nanoconjugates have no absorbance in the visiblespectrum, in contrast to AuNPs that have very strong visible opticalproperties, the system was designed to include fluorescence resonanceenergy transfer (FRET) active fluorophores (Fluorescein (Fl) and Cy3) atthe end of the sequences. Therefore, when the particles hybridize, Flwill transfer energy to Cy3, and the orange fluorescence of Cy3 isobserved. Hollow DNA nanoconjugates were synthesized successfully withthese new strands, and showed similar migration through an agarose gel.Fluorescein can be excited with a UV light source, whereas Cy3 cannot. Asolution of Fl modified particles appeared bright green and the Cy3particles exhibited no typical orange fluorescence. However, when theseparticles were mixed together, the orange fluorescence of Cy3 was easilyvisible under a UV lamp. After time, these particles formed macroscopicaggregates that settled out of solution over time (FIG. 6).Interestingly, when these particles were heated, the bright greenfluorescence of fluorescein was visible, indicating that this processwas reversible. This engineered green to orange color change isanalogous to the red to purple shift evident when DNA-AuNPs aresimilarly hybridized.

That these particles formed macroscopic aggregates over time indicatedthey are binding in a cooperative fashion analogous to DNA-AuNPaggregates. A UV-Vis melting assay was conducted to analyze the degreeof cooperativity between particles. It is well known that the density ofthe DNA on a nanoparticle's surface is directly related to the breadthand temperature of the melting transition of the resulting aggregates[Jin et al., J. Am. Chem. Soc. 125(6): 1643-1654 (2003)].

The extinction of the free strand, DNA-AuNP aggregates and hollow DNAaggregates were monitored at 260 nm of light as a function oftemperature (FIG. 7). The free strands in this system had a meltingtransition at approximately 23° C. When AuNPs were functionalized withthese strands and mixed together, the typical red to purple plasmonicshift occurs, and aggregates were formed [Mirkin et al., Nature382(6592): 607-609 (1996)]. These aggregates melted sharply (full widthat half maximum (FWHM) of the derivative of the melting transition wasapproximately 2° C.) at approximately 43° C. as expected. The aggregatesformed from the hollow nanoconjugates exhibited a similarly sharpmelting transition at approximately 40° C., with the FWHM of thederivative of the melting transition spanning approximately 2° C. Thisextremely similar melting behavior was a direct indication that thepolyvalent melting behavior associated with the DNA-AuNP conjugate waspreserved after crosslinking of the ligand shell and dissolution of thegold core.

Example 6

Having demonstrated that hollow DNA nanoconjugates maintain the size,shape, and function of their DNA-AuNP counterparts, their effectivenessas gene regulation agents was next investigated. RNA hollow particleswere synthesized in the same fashion as DNA hollow particles, but inthis case a DNA/RNA chimera was used, wherein the CR of the strand stillcomprised 10 alkyne-T units, and the CPGs from the synthesis weretransferred to the RNA synthesis to complete the synthesis.Additionally, the antisense RNA strand was labeled with Cy5, so that thehollow particles would be visualized in cells with fluorescencemicroscopy. After dialysis of the hollow particles, the antisensecomplement of the crosslinking strand was added to the hollow particlesin a 100-fold excess to form duplexes on the surface of the particles.HeLa cells were transfected with these particles for 24 hours and imagedwith confocal microscopy. Particles harvested after transfection werevisibly blue as compared to untreated cells, which indicated a very highnumber of particles within the cells.

RNA sequences targeted against EGFR were then synthesized. EGFR is animportant target associated with cancer. SCC12 (human squamouscarcinoma) cells were transfected for various periods of time (48, 72,96 hours) and harvested for their protein and mRNA content. Initialexperiments showed robust knockdown of EGFR normalized to a referencegene, GAPDH (FIG. 8A). Furthermore, western blots showed significantknockdown of EGFR as compared to untreated cells (FIG. 8B).

Example 7

The above examples show that hollow nanoconjugates can be created; next,the nature of the chemistry in this process was investigated. To thatend, multiple model systems were created to investigate the mechanism offormation for these structures. All of these model systems incorporatedalkynes into the ligand shell for crosslinking. A polymer system, asingle alkyne DNA system, and a single alkyne PEG system were designed.

A polymer with alkyne moieties 1 was readily prepared throughpost-polymerization modification of polyacrylamidoethylamine120 with anarrow polydispersity index of 1.10 [Zhang et al., Biomaterials 30 (5),968-977 (2009)]. Containing no charged groups, 1 exhibited moderatesolubility in water at room temperature. Solubility increased at lowertemperatures. Bearing multiple side-arm propargyl ether groups, 1readily adsorbed onto citrate-stabilized 13 nm AuNPs prepared in anaqueous solution by the Turkevich-Frens method (See Scheme 1). Excesspolymers were removed by multiple centrifugation-resuspension steps. Theresulting polymer-coated AuNP retained the plasmon resonance at 524 nmand was stable for months, in contrast to the unmodified poly-aminepolymer, which crashes the particles instantaneously under the sameconditions.

Dissolution of the AuNP core was achieved using 1 mM KCN in the presenceof oxygen. When KCN was added to citrate stabilized AuNPs, the color ofthe solution instantly changed from red to purple, resulting from thedestabilization and aggregation of the AuNPs. However, for thepolymer-coated AuNP, the color slowly changed to a slightly reddishorange color during the dissolution process until the solution was clear(FIG. 9A). This observation suggested that 1 formed a dense crosslinkedshell, which prevented the typical aggregation that is typical of AuNPsbeing oxidatively dissolved. Furthermore, UV-Vis spectra showed agradual decrease of the plasmon resonance from 524 nm to 517 nm (FIG.9B), as expected from the decrease of AuNP size.

The dissolution process was visualized by transmission electronmicroscopy (TEM) (FIG. 9C). As the outer layer of the AuNP was partiallydissolved, the protective shell mentioned above was observed withuranyl-acetate staining of the TEM grid. Complete removal of thetemplate afforded hollow nanoconjugates that retained the size and shapeof their template in high fidelity. When AuNP templates over a range ofsizes were used (10, 20, 30 and 40 nm), the sizes of the resultingpolymer nanoconjugates (PNSs) were directly related to the size of theoriginal template as measured by dynamic light scattering (DLS) and TEM(FIG. 10). These results indicated that the AuNP can serve not only asthe template but also the catalyst for the formation of the PNSs throughthe alkyne moieties. However, they raised the question as to what kindof chemical pathway was accessed in the transformation of 1 to theresulting PNS.

Example 8

IR spectroscopy of 1 before and after crosslinking on the surface of 13nm AuNPs showed the complete loss of the acetylene C≡C stretching (2114cm-1), C—H stretching (3805 cm-1) and bending (1274, 646 cm-1)vibrations, indicating that the propargyl ether group was involved inthe reaction. NMR spectroscopy of the PNSs made from 13 nm AuNP was notpossible, perhaps due to reduced solvent accessibility to the relativelylarge structures (FIG. 11D).

However, 5 nm AuNPs produced PNSs that were suitable for analysis. 1Hand 13C NMR spectroscopy showed the loss of resonances from thepropargyl group, which is likely to leave behind a hydroxyl groupthrough elimination of acrylaldehyde (FIG. 11B) [Fukuda et al., Bull.Chem. Soc. Jpn. 64: 2013-2015 (1991)]. The resulting hydroxyl groupcould then serve as a new nucleophile for remaining alkyne-Au complexesto generate acetal linkages [Fukuda et al., J. Org. Chem. 56(11):3729-3731 (1991)]. Indeed, 1H-13C HSQC spectra showed the appearance ofresonances from the α-H of primary alcohol (δ 3.81 ppm, 1H), alkyl ether(δ 3.74 ppm, 1H) and acetal (δ 4.32 ppm, 1H) groups. Suchtransformations are previously known to be possible only with ionic goldcatalysts. Without being bound by theory, a plausible pathway can beproposed (FIG. 17).

Coordination of the alkynophilic AuNP to 1 is followed by thenucleophilic addition of water to yield intermediate 2, which forms 3 byelimination. Because of high local concentrations on the surface of theAuNP, reaction between the hydroxyl groups and Au-alkyne complexes ispossible without high temperatures, giving acetal and ether crosslinks 4with regeneration of the catalytic AuNP surface sites. However, the AuNPcannot catalyze further reactions because the dense shell that formsshould prevent access of free polymers. Indeed, the polymers remainingin solution were found to be exclusively the starting material 1.Furthermore, this pathway also indicates that a single alkyne moiety canform a 3-arm crosslink.

To support the proposed mechanism, a monodisperse poly(ethyleneglycol)24-propargyl ether conjugate 5 was synthesized, which allowed forconvenient mass and NMR analyses (FIG. 11B). Upon incubation with AuNP,5 was converted to a primary alcohol quantitatively as evidenced bymatrix-assisted laser desorption/ionization time-of-flight massspectrometry (MALDI-TOF MS) and NMR spectroscopy (FIG. 12). Todemonstrate that the chemistry is compatible with complex moleculescontaining various functional groups, a DNA-derivatized alkyne 6 wassynthesized, and incubated with AuNP. Again, 6 was found to lose 38 massunits to give a primary alcohol. More interestingly, from agarose gelelectrophoresis and MALDI-TOF MS, dimers and trimers were observed (FIG.13).

Example 9

Next the polymer that was synthesized was utilized in a functional modelsystem. Functional proteins can be incorporated into the outer shell ofthe polymer nanoconjugates by co-forming the shell on the AuNP templatewith polyvalent propargyl ether 1 (FIG. 14A). Such constructs are termedproteonanoconjugates. As a demonstration, streptavidin and horseradishperoxidase (HRP) were used as model proteins. Streptavidin can bind tosurfaces with tethered biotin moieties, thereby immobilizing theproteonanoconjugate. If present, HRP can then provide a means of readoutby catalyzing a chromogenic reaction between tetramethylbenzidine (TMB)and hydrogen peroxide. In principle, only when both proteins are presentin the proteonanoconjugates, and when both proteins remain active afterthe AuNP dissolution process, will a signal be observed (FIG. 14B).Indeed, signal was only observed when streptavidin, HRP and polymer 1were used in the creation of the proteonanoconjugate shell (FIG. 15).Absence of either HRP or polymer 1 rendered the system non-functional.

Example 10

The present disclosure provides, in various aspects, methods forcrosslinking polynucleotides on a nanoconjugate. This example providesadditional methods for effecting the crosslinking. As described above,the additional methods include DSC and SAC.

In a typical experiment, 1 O.D. of DNA is added to 1 mL of 13 nm goldnanoparticles at a concentration of approximately 10 nM. Polysorbate 20(Tween-20) and phosphate buffer (pH 7.4) are then added to thenanoparticles for a final concentration of 0.01% and 50 mM,respectively. Because the polynucleotides must be as close as possiblefor crosslinking, the nanoparticles are brought up to a high sodiumchloride concentration of 1M to maximize loading. The particles are thencentrifuged (13.2 k rpm) and resuspended in PBS/SDS three times toremove excess DNA.

The crosslinking step is performed by the slow addition of Sulfo-EGS toa final concentration of 1 μM. A slow addition is necessary to preventinterparticle crosslinking, and also to prevent saturation of the DNAstrands with crosslinker, which would result in no crosslinking. Thesolution retains its bright red color, and no aggregation is observed.The particles are then purified by centrifugation (3 times at 13.2 krpm) and are ready to be dissolved.

To dissolve the gold core, potassium cyanide is added to thenanoparticle solution. As the particles dissolve, the bright red colorof the solution fades completely, resulting in a clear solution.Interestingly, in comparison to particles that have been functionalizedwith the amine-modified strand but have not been cross-linked, thecross-linked particles take a significantly longer time to dissolve.Non-crosslinked particles are dissolved within a minute, but thecrosslinked particles can take up to 10 minutes to completely dissolveeven with some light heating. This same effect has been observedelsewhere (Langmuir, 2008, 24 (19), pp 11169-11174), which is evidencefor a cross-linked structure.

To further test the properties of the hollow structures, the overallcoulombic charges present at the surface were analyzed with zetapotential measurements. Gold nanoparticle-DNA conjugates are highlynegatively charged due to the tight arrangement of negatively chargedDNA strands on the surface. The hollow structures should maintain thisproperty if the crosslinking is effective. Three samples were analyzed:DNA-nanoparticle conjugates, dissolved DNA-nanoparticle conjugates thathad not been cross-linked, and dissolved DNA-nanoparticles that havebeen crosslinked—hollow structures. As expected, the dissolved particlesexhibited zeta potential measurements that were nearly identical to andwithin error of the measurements for pure gold nanoparticle-DNAconjugates as represented in Table 4, below.

TABLE 4 Zeta Potential Measurements Non Crosslinked Crosslinked AuNP-DNADissolved Dissolved −38 ± 3 mV −5 ± 3 mV −36 ± 4 mV

Finally, the structural properties of the hollow structures were testedby gel-electrophoresis. In electrophoretic analysis, one can gatherinformation concerning both the size and charge of a molecule ornanoconjugate. Using a 2% agarose gel with ethidium bromide stain at120V, the movement of DNA from dissolved uncrosslinked and crosslinked(hollow) particles was compared. The DNA from the crosslinked structuresmoved faster than the free strands liberated from the uncrosslinkedparticles. This can be explained by the fact that supercoiled DNA, whichcan be in a spherical shape like a hollow particle, travels faster thanfree strand DNA of a smaller size through a gel.

In an additional approach, polynucleotides are synthesized bearing twodistinct polynucleotide nanoconjugates. The first is a nucleotidesequence specific to a target. The other region is a spacer regionharboring a number of pendent alkylamines. These amines are used assynthetic handles to crosslink the polynucleotides with an aminereactive homobifunctional linker, as shown in Scheme 2 (below).Alternatively, these amines can be converted to azides and alkynes viaNHS esters bearing those groups. In that case, “click” chemistry can beused to crosslink the polynucleotides. An additional approach tosynthesizing a dense polynucleotide nanoconjugate without goldnanoparticles is to conjugate polynucleotides to the surface of anpoly(amidoamine) (PAMAM) dendrimer. Polynucleotides functionalized witha terminal decanoic acid moiety are activated with EDC and NHS, and aremixed with PAMAM dendrimers. Alternatively, PAMAM dendrimers terminatedin carboxylate groups are activated with EDC and NHS, and reacted withamine-terminated polynucleotides.

The polynucleotide nanoconjugate is characterized with gelelectrophoresis to measure its mass and polydispersity. Next, thecooperative properties of the polynucleotide nanoconjugate areinvestigated with melting experiments, enzymatic assays, and cellularstudies. In the melting studies, two kinds of polynucleotidenanoconjugate are synthesized with complementary sequences. Afterannealing, the polynucleotide nanoconjugate is melted and the meltingtemperature profile is compared to that of the same free polynucleotidesequences. The polynucleotide nanoconjugates have sharp and elevatedmelting transitions versus free polynucleotides. The cooperativeproperties of the polynucleotide nanoconjugates are investigated withnuclease assays. In these experiments, the rate of strand degradation ismeasured and compared for nanoconjugate and free polynucleotides.

All templated systems designed in this manner resist enzymaticdegradation when compared to free polynucleotides. Cellular uptakestudies are performed to examine enhancement of endocytosis for thesestructures. In addition, gene knockdown studies are performed to measurethe ability of the structures to regulate protein expression.

The present example provides additional methods to, in one aspect,obtain hollow and core-less polynucleotide nanoconjugates through theuse of templated assembly on a nanoparticle surface, crosslinking, andhomobifunctional crosslinkers. These matters are useful in generegulation methods directed to intracellular targets through the use ofboth DNA and siRNA strategies.

What is claimed is:
 1. A composition comprising a nanoconjugate, thenanoconjugate having a defined structure and comprising a plurality ofpolynucleotides crosslinked to each other in a monolayer, said structuredefined by a surface providing a template upon which the structure isassembled.
 2. The composition of claim 1 wherein the surface is ananoparticle.
 3. The composition of claim 1 wherein each polynucleotidein the plurality of polynucleotides is the same.
 4. The composition ofclaim 1 wherein the composition comprises a plurality of nanoconjugates,and the plurality of polynucleotides is monodisperse.
 5. The compositionof claim 4 wherein the monodispersity is such that there is about 25%variation in the diameter of the plurality of nanoconjugates.
 6. Thecomposition of claim 1 wherein the nanoconjugate further comprises anadditional agent selected from the group consisting of a polynucleotide,peptide, polypeptide, phospholipid, oligosaccharide, metal complex,small molecule, therapeutic agent, contrast agent and combinationsthereof.
 7. The composition of claim 6 wherein the additional agent isassociated with at least one polynucleotide of the plurality ofpolynucleotides.
 8. The composition of claim 7 wherein the additionalagent is associated with at least one biomolecule polynucleotide of theplurality of polynucleotides through hybridization, or the additionalagent is covalently associated with at least one biomolecule of theplurality of biomolecules, or the additional agent is entrapped in thecrosslinked biomolecules of the nanoconjugate.
 9. A method ofcrosslinking a structured nanoconjugate, the method comprising the stepof activating a first biomolecule which is a polynucleotide bycontacting the first biomolecule with a surface, the activation allowingthe first biomolecule to crosslink to a second biomolecule.
 10. Themethod of claim 9 wherein the surface provides a template upon which thestructure is assembled.
 11. The method of claim 9 wherein the surface isa nanoparticle.
 12. The method of claim 9 wherein the surface is atleast partially removed after the crosslinking.
 13. The method of claim9 wherein the second biomolecule is selected from the group consistingof a polynucleotide, peptide, polypeptide, phospholipid,oligosaccharide, small molecule, therapeutic agent, contrast agent andcombinations thereof.
 14. The method of claim 9 wherein the firstbiomolecule and the second biomolecule each comprise at least one alkynemoiety, or wherein the first biomolecule and the second biomolecule eachcomprise about 10 alkyne moieties.
 15. The method of claim 14 whereinthe alkyne moiety is activated upon contact with the surface.
 16. Themethod of claim 15 wherein the activation renders the alkyne susceptibleto a nucleophile.
 17. The method of claim 15 wherein activation causescrosslinking of the first biomolecule to the second biomolecule.
 18. Themethod of claim 9 wherein the first biomolecule and the secondbiomolecule are a a DNA polynucleotide or a RNA polynucleotide.
 19. Themethod of claim 9 wherein an additional agent is added to the firstbiomolecule and the second biomolecule during the activating step, orwherein an additional agent is added to the nanoconjugate afterformation of the nanoconjugate but before the at least partial removalof the surface or wherein an additional agent is added to thenanoconjugate after formation of the nanoconjugate and after the atleast partial removal of the surface.
 20. A composition comprising apolyvalent nanoconjugate comprising a surface, the nanoconjugate furthercomprising a plurality of polynucleotides wherein a spacer end of eachof the polynucleotides in the plurality is modified such that contactingthe plurality of polynucleotides with a chemical crosslinks theplurality of polynucleotides to each other.
 21. The composition of claim20 wherein the modification is selected from the group consisting of anamine, amide, alcohol, ester, aldehyde, ketone, thiol, disulfide,carboxylic acid, phenol, imidazole, hydrazine, hydrazone, azide and analkyne.
 22. The composition of claim 20 wherein the surface comprises ananoparticle.
 23. A method of detecting a target molecule comprisingcontacting the target molecule with the composition of claim 1, whereincontact between the target molecule and the composition results in adetectable change.
 24. The method of claim 23 wherein the detecting isin vitro.
 25. A method of inhibiting expression of a gene productencoded by a target polynucleotide comprising contacting the targetpolynucleotide with a composition of claim 1 under conditions sufficientto inhibit expression of the gene product.
 26. The method of claim 25wherein expression of the gene product is inhibited in vivo.
 27. Thecomposition of claim 4 wherein the monodispersity is such that there isabout 10% variation in the diameter of the plurality of nanoconjugates.28. The composition of claim 4 wherein the monodispersity is such thatthere is about 1% variation in the diameter of the plurality ofnanoconjugates.
 29. The composition of claim 1, wherein the surface isat least partially removed after the structure has been defined.
 30. Thecomposition of claim 29 wherein the surface is a nanoparticle.
 31. Thecomposition of claim 29 wherein each polynucleotide in the plurality ofpolynucleotides is the same.
 32. The composition of claim 29 wherein thenanoconjugate further comprises an additional agent selected from thegroup consisting of a polynucleotide, peptide, polypeptide,phospholipid, oligosaccharide, metal complex, small molecule,therapeutic agent, contrast agent and combinations thereof.
 33. Thecomposition of claim 29 wherein the additional agent is associated withat least one polynucleotide of the plurality of polynucleotides.
 34. Thecomposition of claim 29 wherein the nanoconjugate is hollow in theabsence of the surface.
 35. The composition of claim 34 wherein anadditional agent is encapsulated in the nanoconjugate which is otherwisehollow.
 36. The composition of claim 20 wherein the surface is at leastpartially removed after the crosslinking.
 37. The composition of claim36 wherein the modification is selected from the group consisting of anamine, amide, alcohol, ester, aldehyde, ketone, thiol, disulfide,carboxylic acid, phenol, imidazole, hydrazine, hydrazone, azide and analkyne.
 38. The method of claim 23 wherein the detecting is in vivo. 39.The method of claim 25 wherein expression of the gene product isinhibited in vitro.
 40. The composition of claim 1 wherein thepolynucleotide is selected from the group consisting of DNA and RNA. 41.The composition of claim 40 wherein the DNA is double stranded.
 42. Thecomposition of claim 40 wherein the RNA is small interfering RNA(siRNA).
 43. The composition of claim 20 wherein the polynucleotide isselected from the group consisting of DNA and RNA.
 44. The compositionof claim 43 wherein the DNA is double stranded.
 45. The composition ofclaim 43 wherein the RNA is small interfering RNA (siRNA).